IPA® /
DOUBLE IPA®

Integrated Patch Amplifier
ELECTROPHYSIOLOGY
PATCH-CLAMP SYSTEM
WITH
SutterPatch® SOFTWARE

Operation Manual
Rev. 1.1.0 (2017-1-16)

One Digital Drive

Novato, CA 94949 USA

Voice: +1 415-883-0128 Web: www.sutter.com

Fax: +1 415-883-0572 Email: info@sutter.com

The IPA and Double IPA systems have two international certifications:

1) CE marking for compliance to health, safety and environmental protection standards of products sold within the European Economic Area.

2) RoHS directive 2002/95/EC compliance restricting the use of hazardous substances for electronic equipment sold in the European Union:

RoHS-logo-F2696E1677-seeklogo_com(cropped)

IPA^®, Double IPA^®, and SutterPatch^® are trademarks of Sutter Instrument Company.

Other brand names mentioned may be trademarks of other companies.

DISCLAIMER

The IPA system consists of one electronic amplifier with integrated digitizer and one headstage. The Double IPA system consists of one electronic amplifier with integrated digitizer and two headstages. All references to an IPA system also include a Double IPA system, unless otherwise noted. The purpose of the system is for the stimulation and measurement of cellular preparations. No other use is recommended.

This instrument is designed for use in a laboratory environment. It is not intended for, nor should it be used in human experimentation or applied to humans in any way. This is not a medical device.

Do not open or attempt to repair the instrument.

Do not allow an unauthorized and/or untrained operative to use this instrument.

Any misuse will be the sole responsibility of the user/owner, and Sutter Instrument Company assumes no implied or inferred liability for direct or consequential damages from this instrument if it is operated or used in any way other than for which it is designed.

SAFETY WARNINGS AND PRECAUTIONS

Electrical

§ Operate the IPA system using 88 – 264 VAC, 50 - 60 Hz line voltage. This instrument is designed for use in a laboratory environment that has low electromagnetic noise and mechanical vibration. Surge suppression is recommended at all times.

Fuse Replacement: Replace only with the same type and rating:

Line Voltage: 88 – 264 VAC

Fuse

Rating

Manufacturer Examples

RoHS Compliant (Lead Free)

T2.0,

250V

Bussmann: GMC-2-R, S506-2A
Littelfuse: 239.002.P

Type: 5 x 20 mm glass tube, Medium Time Delay (Slow Blow), RoHS compliant.

Rating: T2.0A 250V (Time Delay, 2 Amps, 250 Volts)

Examples: Bussmann: GMC-2-R, S506-2A
Littelfuse: 239.002.P

§ Avoiding Electrical Shock and Fire-related Injury

§ Always use the grounded power cord provided to connect the system’s power adapter to a grounded/earthed mains outlet. This is required to protect you from injury in the event that an electrical hazard occurs.

§ Do not disassemble the system. Refer servicing to qualified personnel.

§ To prevent fire or shock hazard, do not expose the unit to rain or moisture.

Operational

Failure to comply with any of the following precautions may damage this instrument.

§ This instrument is designed for operation in a laboratory environment (Pollution Degree I) that is free from mechanical vibrations, electrical noise and transients.

§ Operate this instrument only according to the instructions included in this manual.

§ Do not operate this instrument near flammable materials. The use of any hazardous materials with this instrument is not recommended and, if undertaken, is done so at the users’ own risk.

§ Do not operate if there is any obvious damage to any part of the instrument.

Other

§ Retain the original packaging for future transport of the instrument.

§ Sutter Instrument Company reserves the right to change specifications without prior notice.

§ Use of this instrument is for research purposes only.

Handling Micropipettes

Failure to comply with any of the following precautions may result in injury to the users of this instrument as well as those working in the general area near the instrument.

§ The micropipettes used with this instrument are very sharp and relatively fragile. Avoid contact with micropipette tips to prevent accidentally impaling yourself.

§ Always dispose of micropipettes by placing them into a well-marked, spill-proof “sharps” container.

TABLE OF CONTENTS

DISCLAIMER............................................................................................................................. 3

SAFETY WARNINGS AND PRECAUTIONS................................................................... 3

Electrical.................................................................................................................................... 3

Operational................................................................................................................................ 4

Other........................................................................................................................................... 4

Handling Micropipettes.......................................................................................................... 4

1. INTRODUCTION............................................................................................................... 13

1.1 Overview............................................................................................................................ 13

1.2 Software Highlights......................................................................................................... 14

1.3 Experiment Structure..................................................................................................... 14

2. INSTALLATION................................................................................................................. 17

2.1 Computer Requirements................................................................................................ 17

2.2 SutterPatch System Environment................................................................................ 18

2.3 Mounting Instructions.................................................................................................... 19

2.4 Electrical Connections.................................................................................................... 19

2.5 Install Hardware.............................................................................................................. 20

2.6 Install Software................................................................................................................ 21

2.7 Test System....................................................................................................................... 21

2.7.1 Install Model Cell....................................................................................................................................................................... 21

2.7.2 Startup............................................................................................................................................................................................. 21

2.7.3 Run a Membrane Test............................................................................................................................................................ 24

3. HARDWARE OPERATION............................................................................................. 26

3.1 IPA Front Panel................................................................................................................ 26

3.2 Double IPA Front Panel.................................................................................................. 29

3.3 IPA Rear Panel................................................................................................................. 29

3.4 Grounding............................................................................................................................ 30

3.5 Headstage............................................................................................................................. 30

3.6 Holder.................................................................................................................................. 30

3.6.1 Assembly........................................................................................................................................................................................ 31

3.6.2 Chloriding Silver Wire............................................................................................................................................................ 32

3.6.3 Holder Maintenance................................................................................................................................................................ 33

3.7 Maintenance......................................................................................................................... 34

3.7.1 Inspection...................................................................................................................................................................................... 34

3.7.2 Cleaning.......................................................................................................................................................................................... 34

3.8 Calibration............................................................................................................................ 34

3.9 Amplifier Control Panel (Software)............................................................................. 35

3.10 Using Peripheral Equipment....................................................................................... 43

3.11 Using Multiple Sutter Amplifiers...................................................................................... 44

3.12 Using Non-Sutter Amplifiers....................................................................................... 44

3.13 Using Non-Sutter Data Acquisition Systems............................................................ 44

4. SOFTWARE OPERATION.............................................................................................. 45

4.1 Acquisition........................................................................................................................ 45

4.1.1 Camera Module.......................................................................................................................................................................... 46

4.1.2 Free Run......................................................................................................................................................................................... 47

4.1.3 Membrane Test.......................................................................................................................................................................... 47

4.1.4 Paradigm Editor......................................................................................................................................................................... 48

Amplifier................................................................................................................................................................................................. 55

Each Sweep............................................................................................................................................................................................ 60

Routine.................................................................................................................................................................................................... 60

Analysis................................................................................................................................................................................................... 61

Camera..................................................................................................................................................................................................... 61

Clear Key................................................................................................................................................................................................. 62

Execute.................................................................................................................................................................................................... 62

Export...................................................................................................................................................................................................... 64

Front Window....................................................................................................................................................................................... 65

Hide Window........................................................................................................................................................................................ 66

Reset Timer............................................................................................................................................................................................ 66

Scope Operation................................................................................................................................................................................... 67

Set Axis.................................................................................................................................................................................................... 67

Set Checkbox......................................................................................................................................................................................... 68

Set Solution............................................................................................................................................................................................ 69

Set Tag..................................................................................................................................................................................................... 70

Set Variable............................................................................................................................................................................................ 70

Sound....................................................................................................................................................................................................... 72

View Last................................................................................................................................................................................................. 73

Write Log................................................................................................................................................................................................ 73

Alert.......................................................................................................................................................................................................... 74

Beep.......................................................................................................................................................................................................... 75

Comment................................................................................................................................................................................................ 75

Break........................................................................................................................................................................................................ 76

Chain......................................................................................................................................................................................................... 76

For Loop.................................................................................................................................................................................................. 77

Jump.......................................................................................................................................................................................................... 77

Label......................................................................................................................................................................................................... 77

If................................................................................................................................................................................................................. 78

ElseIf......................................................................................................................................................................................................... 80

Else............................................................................................................................................................................................................ 82

4.1.5 (Scope) Real Time Measurements & Graphs............................................................................................................. 87

4.1.6 Routine Editor............................................................................................................................................................................. 89

Routine Editor: Acquisition & Routine Parameters................................................................................................................ 95

Routine Editor: Input Channels...................................................................................................................................................... 98

Routine Editor: Output Channels & Waveform..................................................................................................................... 104

Routine Editor: Waveform Editor.............................................................................................................................................. 110

Routine Editor: Real Time Measurements & Graphs.......................................................................................................... 118

Routine Editor: Routine Variables............................................................................................................................................ 123

4.1.7 Scope (Acquisition)............................................................................................................................................................... 124

4.1.8 Solution Editor......................................................................................................................................................................... 134

4.1.9 Template Editor...................................................................................................................................................................... 135

4.2 Analysis.............................................................................................................................. 138

4.2.1 Action Potential Analysis................................................................................................................................................... 138

4.2.2 Analysis Editor......................................................................................................................................................................... 142

4.2.3 Analysis Window................................................................................................................................................................... 147

4.2.4 Data Browser............................................................................................................................................................................ 148

4.2.5 Data Navigator......................................................................................................................................................................... 150

4.2.6 Data Table.................................................................................................................................................................................. 154

4.2.7 Equation Editor....................................................................................................................................................................... 155

4.2.8 Event Detection....................................................................................................................................................................... 163

4.2.9 Igor Analyses............................................................................................................................................................................ 165

4.2.10 Layout Window.................................................................................................................................................................... 167

4.2.11 Metadata Review................................................................................................................................................................. 167

4.2.12 Paradigm Review................................................................................................................................................................ 171

4.2.13 Reanalysis Measurements & Graphs........................................................................................................................ 171

4.2.14 Routine Review.................................................................................................................................................................... 177

4.2.15 Scope (Reanalysis)............................................................................................................................................................. 179

4.2.16 3D View Window................................................................................................................................................................ 181

4.2.17 Set Metadata........................................................................................................................................................................... 183

4.3 General............................................................................................................................ 195

4.3.1 Command Window............................................................................................................................................................... 195

4.3.2 Dashboard Panel.................................................................................................................................................................... 195

4.3.3 File Import/Export................................................................................................................................................................ 196

4.3.4 Log Window.............................................................................................................................................................................. 198

4.3.5 Menus........................................................................................................................................................................................... 200

4.3.6 Preferences................................................................................................................................................................................ 204

i. General....................................................................................................................................................................................... 205

ii. Files and Naming.................................................................................................................................................................... 206

iii. Scope...................................................................................................................................................................................... 206

iv. Membrane Test............................................................................................................................................................... 209

v. Export Graphics....................................................................................................................................................................... 209

vi. Hardware.............................................................................................................................................................................. 211

vii. Metadata............................................................................................................................................................................. 212

viii. Factory Reset....................................................................................................................................................................... 212

4.3.7 Shortcut Editor........................................................................................................................................................................ 212

4.3.8 Sample Files.............................................................................................................................................................................. 217

4.3.9 Startup.......................................................................................................................................................................................... 219

5. Programming.................................................................................................................... 220

5.1 Data Format....................................................................................................................... 220

5.2 Data Structure.................................................................................................................... 220

5.3 Data Paths.......................................................................................................................... 220

5.4 User Functions................................................................................................................... 221

6. MAINTENANCE............................................................................................................... 221

6.1 Inspection.......................................................................................................................... 221

6.2 Cleaning............................................................................................................................. 222

6.3 Calibration......................................................................................................................... 222

7. TROUBLESHOOTING................................................................................................... 222

7.1 Technical Support.............................................................................................................. 222

7.2 Startup Issues..................................................................................................................... 223

7.2.1 Installation Fails...................................................................................................................................................................... 223

7.2.2 Application Not Loading.................................................................................................................................................... 223

7.2.3 Startup Errors.......................................................................................................................................................................... 223

7.3 Acquisition Issues.......................................................................................................... 223

7.3.1 Acquisition Locked................................................................................................................................................................ 223

7.3.2 Channel Saturated................................................................................................................................................................. 223

7.3.3 Headstage Noise I................................................................................................................................................................... 223

7.3.4 Headstage Noise II................................................................................................................................................................. 224

7.3.5 Amplifier Output Attenuation......................................................................................................................................... 224

7.3.6 Paradigm Sound Reduced................................................................................................................................................. 225

7.3.7 Offset Zero Delay.................................................................................................................................................................... 225

7.3.8 Metadata Written................................................................................................................................................................... 225

7.3.9 Post-Acquisition Delay........................................................................................................................................................ 225

7.3.10 Sluggish Acquisition.......................................................................................................................................................... 225

7.4 Analysis Issues................................................................................................................... 225

7.4.1 Analysis Not Deleted............................................................................................................................................................ 225

7.4.2 Signal Axes Overlay............................................................................................................................................................... 226

7.4.3 Graphs & Layouts Not Visible.......................................................................................................................................... 226

7.5 General Issues................................................................................................................ 226

7.5.1 Slow Computer........................................................................................................................................................................ 226

7.5.2 Slow Sweeps............................................................................................................................................................................. 226

7.5.3 Window Maximizing............................................................................................................................................................ 226

7.5.4 Command Window Frozen.............................................................................................................................................. 227

7.5.5 Strange Characters................................................................................................................................................................ 227

7.5.6 Wrong Default Settings....................................................................................................................................................... 227

7.5.7 Weird Behavior....................................................................................................................................................................... 227

7.5.8 Igor Pro Features.................................................................................................................................................................... 227

APPENDIX A: LIMITED WARRANTY.......................................................................... 228

APPENDIX B: SOFTWARE LICENSE.......................................................................... 228

APPENDIX C: ACCESSORIES........................................................................................ 234

APPENDIX D: FUSE REPLACEMENT........................................................................ 234

APPENDIX E. TECHNICAL SPECIFICATIONS....................................................... 236

INDEX...................................................................................................................................... 241

~~DISCLAIMER............................................................................................................................. 3~~

~~SAFETY WARNINGS AND PRECAUTIONS................................................................... 3~~

~~Electrical.................................................................................................................................... 3~~

~~Operational................................................................................................................................ 4~~

~~Other........................................................................................................................................... 4~~

~~Handling Micropipettes.......................................................................................................... 4~~

~~1. INTRODUCTION............................................................................................................... 13~~

~~1.1 Overview............................................................................................................................ 13~~

~~1.2 Software Highlights......................................................................................................... 14~~

~~1.3 Experiment Structure..................................................................................................... 14~~

~~2. INSTALLATION................................................................................................................. 17~~

~~2.1 Computer Requirements................................................................................................ 17~~

~~2.2 SutterPatch System Environment................................................................................ 18~~

~~2.3 Mounting Instructions.................................................................................................... 19~~

~~2.4~~ ~~Electrical Connections.................................................................................................... 19~~

~~2.5 Install Hardware.............................................................................................................. 20~~

~~2.6 Install Software................................................................................................................ 21~~

~~2.7 Test System....................................................................................................................... 21~~

~~2.7.1 Install Model Cell....................................................................................................................................................................... 21~~

~~2.7.3 Run a Membrane Test............................................................................................................................................................ 24~~

~~3. HARDWARE OPERATION............................................................................................. 26~~

~~3.1 IPA Front Panel................................................................................................................ 26~~

~~3.2 Double IPA Front Panel.................................................................................................. 29~~

~~3.3 IPA Rear Panel................................................................................................................. 29~~

~~3.4 Grounding............................................................................................................................ 30~~

~~3.5 Headstage............................................................................................................................. 30~~

~~3.6 Holder.................................................................................................................................. 30~~

~~3.6.2 Chloriding Silver Wire............................................................................................................................................................ 32~~

~~3.6.3 Holder Maintenance................................................................................................................................................................ 33~~

~~3.7 Maintenance......................................................................................................................... 34~~

~~3.8 Calibration............................................................................................................................ 34~~

~~3.9 Amplifier Control Panel (Software)............................................................................. 35~~

~~3.10 Using Peripheral Equipment....................................................................................... 43~~

~~3.11~~ ~~Using~~ ~~Multiple Sutter Amplifiers...................................................................................... 44~~

~~3.12 Using Non-Sutter Amplifiers....................................................................................... 44~~

~~3.13 Using Non-Sutter Data Acquisition Systems............................................................ 44~~

~~4. SOFTWARE OPERATION.............................................................................................. 45~~

~~4.1 Acquisition........................................................................................................................ 45~~

~~4.1.1 Camera Module.......................................................................................................................................................................... 46~~

~~4.1.3 Membrane Test.......................................................................................................................................................................... 47~~

~~4.1.4 Paradigm Editor......................................................................................................................................................................... 49~~

~~Front Window....................................................................................................................................................................................... 65~~

~~Hide Window........................................................................................................................................................................................ 66~~

~~Scope Operation................................................................................................................................................................................... 67~~

~~Set Checkbox......................................................................................................................................................................................... 68~~

~~4.1.5 (Scope) Real Time Measurements & Graphs............................................................................................................. 87~~

~~4.1.6 Routine Editor............................................................................................................................................................................. 89~~

~~Routine Editor: Acquisition & Routine Parameters................................................................................................................ 95~~

~~Routine Editor: Input Channels...................................................................................................................................................... 98~~

~~Routine Editor: Output Channels & Waveform..................................................................................................................... 104~~

~~Routine Editor: Waveform Editor.............................................................................................................................................. 111~~

~~Routine Editor: Real Time Measurements & Graphs.......................................................................................................... 119~~

~~Routine Editor: Routine Variables............................................................................................................................................ 123~~

~~4.1.7 Scope (Acquisition)............................................................................................................................................................... 124~~

~~4.1.8 Solution Editor......................................................................................................................................................................... 134~~

~~4.1.9 Template Editor...................................................................................................................................................................... 135~~

~~4.2 Analysis.............................................................................................................................. 138~~

~~4.2.1 Action Potential Analysis................................................................................................................................................... 138~~

~~4.2.2 Analysis Editor......................................................................................................................................................................... 142~~

~~4.2.3 Analysis Window................................................................................................................................................................... 147~~

~~4.2.4 Data Browser............................................................................................................................................................................ 148~~

~~4.2.5 Data Navigator......................................................................................................................................................................... 150~~

~~4.2.6 Data Table.................................................................................................................................................................................. 154~~

~~4.2.7 Equation Editor....................................................................................................................................................................... 155~~

~~4.2.8 Event Detection....................................................................................................................................................................... 163~~

~~4.2.9 Igor Analyses............................................................................................................................................................................ 165~~

~~4.2.10 Layout Window.................................................................................................................................................................... 166~~

~~4.2.11 Metadata Review................................................................................................................................................................. 167~~

~~4.2.12 Paradigm Review................................................................................................................................................................ 171~~

~~4.2.13 Reanalysis Measurements & Graphs........................................................................................................................ 171~~

~~4.2.14 Routine Review.................................................................................................................................................................... 177~~

~~4.2.15 Scope (Reanalysis)............................................................................................................................................................. 179~~

~~4.2.16 3D View Window................................................................................................................................................................ 181~~

~~4.2.17 Set Metadata........................................................................................................................................................................... 183~~

~~4.3 General............................................................................................................................ 195~~

~~4.3.1 Command Window............................................................................................................................................................... 195~~

~~4.3.2 Dashboard Panel.................................................................................................................................................................... 195~~

~~4.3.3 File Import/Export................................................................................................................................................................ 196~~

~~4.3.4 Log Window.............................................................................................................................................................................. 198~~

~~4.3.6 Preferences................................................................................................................................................................................ 204~~

i. ~~General....................................................................................................................................................................................... 205~~

~~ii.~~ ~~Files and Naming.................................................................................................................................................................... 206~~

~~iii.~~ ~~Scope...................................................................................................................................................................................... 206~~

~~iv.~~ ~~Membrane Test............................................................................................................................................................... 209~~

v. ~~Export Graphics....................................................................................................................................................................... 210~~

~~vi.~~ ~~Hardware.............................................................................................................................................................................. 211~~

~~vii.~~ ~~Metadata............................................................................................................................................................................. 212~~

~~viii.~~ ~~Factory Reset....................................................................................................................................................................... 212~~

~~4.3.7 Shortcut Editor........................................................................................................................................................................ 212~~

~~4.3.8 Sample Files.............................................................................................................................................................................. 217~~

~~5. Programming.................................................................................................................... 220~~

~~5.1 Data Format....................................................................................................................... 220~~

~~5.2 Data Structure.................................................................................................................... 220~~

~~5.3 Data Paths.......................................................................................................................... 221~~

~~5.4 User Functions................................................................................................................... 221~~

~~6. MAINTENANCE............................................................................................................... 222~~

~~6.1 Inspection.......................................................................................................................... 222~~

~~6.2 Cleaning............................................................................................................................. 222~~

~~6.3 Calibration......................................................................................................................... 222~~

~~7. TROUBLESHOOTING................................................................................................... 222~~

~~7.1 Technical Support.............................................................................................................. 222~~

~~7.2 Startup Issues..................................................................................................................... 223~~

~~7.2.1 Installation Fails...................................................................................................................................................................... 223~~

~~7.2.2 Application Not Loading.................................................................................................................................................... 223~~

~~7.2.3 Startup Errors.......................................................................................................................................................................... 224~~

~~7.3 Acquisition Issues.......................................................................................................... 224~~

~~7.3.1 Acquisition Locked................................................................................................................................................................ 224~~

~~7.3.2 Channel Saturated................................................................................................................................................................. 224~~

~~7.3.3 Headstage Noise I................................................................................................................................................................... 224~~

~~7.3.4 Headstage Noise II................................................................................................................................................................. 224~~

~~7.3.5 Amplifier Output Attenuation......................................................................................................................................... 224~~

~~7.3.6 Paradigm Sound Reduced................................................................................................................................................. 225~~

~~7.3.7 Offset Zero Delay.................................................................................................................................................................... 225~~

~~7.3.8 Metadata Written................................................................................................................................................................... 225~~

~~7.3.9 Post-Acquisition Delay........................................................................................................................................................ 225~~

~~7.3.10 Sluggish Acquisition.......................................................................................................................................................... 225~~

~~7.4 Analysis Issues................................................................................................................... 226~~

~~7.4.1 Analysis Not Deleted............................................................................................................................................................ 226~~

~~7.4.2 Signal Axes Overlay............................................................................................................................................................... 226~~

~~7.4.3 Graphs & Layouts Not Visible.......................................................................................................................................... 226~~

~~7.5~~ ~~General Issues................................................................................................................ 226~~

~~7.5.1 Slow Computer........................................................................................................................................................................ 226~~

~~7.5.2 Slow Sweeps............................................................................................................................................................................. 226~~

~~7.5.3 Window Maximizing............................................................................................................................................................ 226~~

~~7.5.4 Command Window Frozen.............................................................................................................................................. 227~~

~~7.5.5 Strange Characters................................................................................................................................................................ 227~~

~~7.5.6 Wrong Default Settings....................................................................................................................................................... 227~~

~~7.5.7 Weird Behavior....................................................................................................................................................................... 227~~

~~7.5.8 Igor Pro Features.................................................................................................................................................................... 227~~

~~APPENDIX A: LIMITED WARRANTY.......................................................................... 228~~

~~APPENDIX B: SOFTWARE LICENSE.......................................................................... 228~~

~~APPENDIX C: ACCESSORIES........................................................................................ 234~~

~~APPENDIX D: FUSE REPLACEMENT........................................................................ 234~~

~~APPENDIX E. TECHNICAL SPECIFICATIONS....................................................... 236~~

~~INDEX...................................................................................................................................... 241~~

TABLE OF FiGURES

Figure 1‑1. SutterPatch Data Structure....................................................................................... 15

Figure 2‑1. Rear of IPA Cabinet.................................................................................................... 20

Figure 2‑2. Front of IPA Cabinet.................................................................................................. 20

Figure 2‑3. Front of Double IPA Cabinet...................................................................................... 20

Figure 2‑4. Splash Screen............................................................................................................. 22

Figure 2‑5: Welcome Screen.......................................................................................................... 22

Figure 2‑6. USB / Emulation Screen............................................................................................. 23

Figure 2‑7. Amplifier Control Panel.............................................................................................. 24

Figure 2‑8. Dashboard................................................................................................................... 25

Figure 2‑9. Dashboard – Acquisition............................................................................................. 25

Figure 3‑1: Front of IPA Cabinet.................................................................................................. 26

Figure 3‑2. Front of Double IPA Cabinet...................................................................................... 29

Figure 3‑3: Rear of IPA Cabinet.................................................................................................... 29

Figure 3‑4: Electrode Holder......................................................................................................... 31

Figure 3‑5: Amplifier Control Panel.............................................................................................. 35

Figure 3‑6. Headstage Monitor..................................................................................................... 43

Figure 4‑1. Paradigm Editor......................................................................................................... 49

Figure 4‑2. Step: Amplifier............................................................................................................ 55

Figure 4‑3. Step: Each Sweep........................................................................................................ 60

Figure 4‑4. Step: Routine.............................................................................................................. 60

Figure 4‑5. Step: Analysis............................................................................................................. 61

Figure 4‑6. Step: Scope Operation................................................................................................. 67

Figure 4‑7. Step: Axis.................................................................................................................... 68

Figure 4‑8. Step: Checkbox........................................................................................................... 69

Figure 4‑9. Step: Set Solution....................................................................................................... 70

Figure 4‑10. Step: Set Tag............................................................................................................. 70

Figure 4‑11. Step: Set Variable..................................................................................................... 71

Figure 4‑12. Step: Sound............................................................................................................... 72

Figure 4‑13. Step: Write Log......................................................................................................... 73

Figure 4‑14. Step: Alert................................................................................................................. 74

Figure 4‑15. Step Comment.......................................................................................................... 75

Figure 4‑16. Step: Wait................................................................................................................. 75

Figure 4‑17. Step: Break............................................................................................................... 76

Figure 4‑18. Step: Chain............................................................................................................... 76

Figure 4‑19. Step: For Loop........................................................................................................... 77

Figure 4‑20. Step: Jump................................................................................................................ 77

Figure 4‑21. Step: Label................................................................................................................ 78

Figure 4‑22. Step: If....................................................................................................................... 78

Figure 4‑23. Step: Else If............................................................................................................... 80

Figure 4‑24. Checkboxes............................................................................................................... 82

Figure 4‑25. Paradigm Variables.................................................................................................. 83

Figure 4‑26. Acquisition Real Time Measurements & Graphs..................................................... 88

Figure 4‑27. Routine Editor.......................................................................................................... 89

Figure 4‑28. New Routine Pool..................................................................................................... 91

Figure 4‑29: Waveform Preview Pane........................................................................................... 93

Figure 4‑30. Routine Settings Sub-Window.................................................................................. 95

Figure 4‑31. Acquisition & Routine Parameters........................................................................... 95

Figure 4‑32. Input Channels......................................................................................................... 99

Figure 4‑33. Output Channels & Waveform............................................................................... 104

Figure 4‑34. Waveform Editor.................................................................................................... 110

Figure 4‑35. Template Waves..................................................................................................... 114

Figure 4‑36. Real Time Measurements & Graphs...................................................................... 119

Figure 4‑37. Routine Variables................................................................................................... 124

Figure 4‑38 Scope Acquisition Window....................................................................................... 125

Figure 4‑39. Signal Display Mode............................................................................................... 126

Figure 4‑40. Axis magnification.................................................................................................. 127

Figure 4‑41. Axis Scroll Bar........................................................................................................ 128

Figure 4‑42. Center Button......................................................................................................... 129

Figure 4‑43. Autoscale All Axes.................................................................................................. 129

Figure 4‑44. Amplitude Meters................................................................................................... 130

Figure 4‑45. Solution Editor........................................................................................................ 134

Figure 4‑46. Template Editor...................................................................................................... 136

Figure 4‑47. Action Potential Analysis....................................................................................... 139

Figure 4‑48. Action Potential Measurements............................................................................. 141

Figure 4‑49. Analysis Editor....................................................................................................... 143

Figure 4‑50. Analysis Window.................................................................................................... 147

Figure 4‑51. Data Browser.......................................................................................................... 149

Figure 4‑52: Data Navigator....................................................................................................... 150

Figure 4‑53. Data Table.............................................................................................................. 154

Figure 4‑54. Equation Editor...................................................................................................... 155

Figure 4‑55. Reanalysis Measurements & Graphs..................................................................... 172

Figure 4‑56: Edit Virtual Signals................................................................................................ 173

Figure 4‑57. Routine Review....................................................................................................... 177

Figure 4‑58. Scope Reanalysis Window...................................................................................... 179

Figure 4‑59. Navigation Pane..................................................................................................... 179

Figure 4‑60. 3D View................................................................................................................... 182

Figure 4‑61. 3D Axis Definition.................................................................................................. 183

Figure 4‑62. SutterPatch Dashboard.......................................................................................... 196

Figure 4‑63. Dashboard – Acquire Data..................................................................................... 196

Figure 4‑64: Preferences Settings............................................................................................... 205

Figure 4‑65: Shortcuts Editor...................................................................................................... 213

Figure E‑0‑1. Electrode Holder................................................................................................... 241

~~Figure 1‑1. SutterPatch Data Structure....................................................................................... 15~~

~~Figure 2‑1. Rear of IPA Cabinet.................................................................................................... 20~~

~~Figure 2‑2. Front of IPA Cabinet.................................................................................................. 20~~

~~Figure 2‑3. Front of Double IPA Cabinet...................................................................................... 20~~

~~Figure 2‑4. Splash Screen............................................................................................................. 22~~

~~Figure 2‑5: Welcome Screen.......................................................................................................... 22~~

~~Figure 2‑6. USB / Emulation Screen............................................................................................. 23~~

~~Figure 2‑7. Amplifier Control Panel.............................................................................................. 24~~

~~Figure 2‑8. Dashboard................................................................................................................... 25~~

~~Figure 2‑9. Dashboard – Acquisition............................................................................................. 25~~

~~Figure 3‑1: Front of IPA Cabinet.................................................................................................. 26~~

~~Figure 3‑2. Front of Double IPA Cabinet...................................................................................... 29~~

~~Figure 3‑3: Rear of IPA Cabinet.................................................................................................... 29~~

~~Figure 3‑4: Electrode Holder......................................................................................................... 31~~

~~Figure 3‑5: Amplifier Control Panel.............................................................................................. 35~~

~~Figure 3‑6. Headstage Monitor..................................................................................................... 43~~

~~Figure 4‑1. Paradigm Editor......................................................................................................... 49~~

~~Figure 4‑2. Step: Amplifier............................................................................................................ 55~~

~~Figure 4‑3. Step: Each Sweep........................................................................................................ 60~~

~~Figure 4‑4. Step: Routine.............................................................................................................. 60~~

~~Figure 4‑5. Step: Analysis............................................................................................................. 61~~

~~Figure 4‑6. Step: Scope Operation................................................................................................. 67~~

~~Figure 4‑7. Step: Axis.................................................................................................................... 68~~

~~Figure 4‑8. Step: Checkbox........................................................................................................... 69~~

~~Figure 4‑9. Step: Set Solution....................................................................................................... 70~~

~~Figure 4‑10. Step: Set Tag............................................................................................................. 70~~

~~Figure 4‑11. Step: Set Variable..................................................................................................... 71~~

~~Figure 4‑12. Step: Sound............................................................................................................... 72~~

~~Figure 4‑13. Step: Write Log......................................................................................................... 73~~

~~Figure 4‑14. Step: Alert................................................................................................................. 74~~

~~Figure 4‑15. Step Comment.......................................................................................................... 75~~

~~Figure 4‑16. Step: Wait................................................................................................................. 75~~

~~Figure 4‑17. Step: Break............................................................................................................... 76~~

~~Figure 4‑18. Step: Chain............................................................................................................... 76~~

~~Figure 4‑19. Step: For Loop........................................................................................................... 77~~

~~Figure 4‑20. Step: Jump................................................................................................................ 77~~

~~Figure 4‑21. Step: Label................................................................................................................ 78~~

~~Figure 4‑22. Step: If....................................................................................................................... 78~~

~~Figure 4‑23. Step: Else If............................................................................................................... 80~~

~~Figure 4‑24. Checkboxes............................................................................................................... 82~~

~~Figure 4‑25. Paradigm Variables.................................................................................................. 83~~

~~Figure 4‑36. Acquisition Real Time Measurements & Graphs..................................................... 88~~

~~Figure 4‑26. Routine Editor.......................................................................................................... 89~~

~~Figure 4‑27. New Routine Pool..................................................................................................... 91~~

~~Figure 4‑28: Waveform Preview Pane........................................................................................... 93~~

~~Figure 4‑29. Routine Settings Sub-Window.................................................................................. 95~~

~~Figure 4‑30. Acquisition & Routine Parameters........................................................................... 95~~

~~Figure 4‑31. Input Channels......................................................................................................... 99~~

~~Figure 4‑32. Output Channels & Waveform............................................................................... 105~~

~~Figure 4‑33. Waveform Editor.................................................................................................... 111~~

~~Figure 4‑34. Template Waves..................................................................................................... 114~~

~~Figure 4‑35. Real Time Measurements & Graphs...................................................................... 119~~

~~Figure 4‑37. Routine Variables................................................................................................... 124~~

~~Figure 4‑38 Scope Acquisition Window....................................................................................... 125~~

~~Figure 4‑39. Signal Display Mode............................................................................................... 126~~

~~Figure 4‑40. Axis magnification.................................................................................................. 127~~

~~Figure 4‑41. Axis Scroll Bar........................................................................................................ 128~~

~~Figure 4‑42. Center Button......................................................................................................... 129~~

~~Figure 4‑43. Autoscale All Axes.................................................................................................. 129~~

~~Figure 4‑44. Amplitude Meters................................................................................................... 130~~

~~Figure 4‑45. Solution Editor........................................................................................................ 134~~

~~Figure 4‑46. Template Editor...................................................................................................... 136~~

~~Figure 4‑47. Action Potential Analysis....................................................................................... 139~~

~~Figure 4‑48. Action Potential Measurements............................................................................. 141~~

~~Figure 4‑49. Analysis Editor....................................................................................................... 143~~

~~Figure 4‑50. Analysis Window.................................................................................................... 147~~

~~Figure 4‑51. Data Browser.......................................................................................................... 149~~

~~Figure 4‑52: Data Navigator....................................................................................................... 150~~

~~Figure 4‑53. Data Table.............................................................................................................. 154~~

~~Figure 4‑54. Equation Editor...................................................................................................... 155~~

~~Figure 4‑55. Reanalysis Measurements & Graphs..................................................................... 172~~

~~Figure 4‑56: Edit Virtual Signals................................................................................................ 173~~

~~Figure 4‑57. Routine Review....................................................................................................... 177~~

~~Figure 4‑58. Scope Reanalysis Window...................................................................................... 179~~

~~Figure 4‑59. Navigation Pane..................................................................................................... 179~~

~~Figure 4‑60. 3D View................................................................................................................... 182~~

~~Figure 4‑61. 3D Axis Definition.................................................................................................. 183~~

~~Figure 4‑62. SutterPatch Dashboard.......................................................................................... 196~~

~~Figure 4‑63. Dashboard – Acquire Data..................................................................................... 196~~

~~Figure 4‑64: Preferences Settings............................................................................................... 205~~

~~Figure 4‑65: Shortcuts Editor...................................................................................................... 213~~

~~Figure E‑0‑1. Electrode Holder................................................................................................... 241~~

TABLE OF TABLES

Table 1‑1: Software Terminology.................................................................................................. 17

Table 4‑1: Files and Pools.............................................................................................................. 90

Table 4‑2. Routine Editor Buttons................................................................................................ 92

Table 4‑3: Other Buttons............................................................................................................. 132

Table 4‑4: Equation Parser......................................................................................................... 161

Table 4‑5: Engineering Notation................................................................................................. 162

Table 4‑6: Scope Window Buttons............................................................................................... 181

Table D‑0‑1: IPA Fuses............................................................................................................... 235

Table E‑0‑1. IPA Amplifier Specifications................................................................................... 237

Table E‑0‑2. IPA Headstage Specifications................................................................................. 238

Table E‑0‑3. IPA Headstage Noise.............................................................................................. 238

Table E‑0‑4. IPA Amplifier Bandwidth....................................................................................... 238

Table E‑0‑5. IPA Digitizer Specifications.................................................................................... 239

Table E‑0‑6. IPA Expansion Panel Specifications....................................................................... 239

Table E‑0‑7. IPA Electrical Specifications.................................................................................. 239

~~Table 1‑1: Software Terminology.................................................................................................. 17~~

~~Table 4‑1: Files and Pools.............................................................................................................. 90~~

~~Table 4‑2. Routine Editor Buttons................................................................................................ 92~~

~~Table 4‑3: Other Buttons............................................................................................................. 132~~

~~Table 4‑4: Equation Parser......................................................................................................... 161~~

~~Table 4‑5: Engineering Notation................................................................................................. 162~~

~~Table 4‑6: Scope Window Buttons............................................................................................... 181~~

~~Table D‑0‑1: IPA Fuses............................................................................................................... 235~~

~~Table E‑0‑1. IPA Amplifier Specifications................................................................................... 237~~

~~Table E‑0‑2. IPA Headstage Specifications................................................................................. 238~~

~~Table E‑0‑3. IPA Headstage Noise.............................................................................................. 238~~

~~Table E‑0‑4. IPA Amplifier Bandwidth....................................................................................... 238~~

~~Table E‑0‑5. IPA Digitizer Specifications.................................................................................... 239~~

~~Table E‑0‑6. IPA Expansion Panel Specifications....................................................................... 239~~

~~Table E‑0‑7. IPA Electrical Specifications.................................................................................. 239~~

1.2 Software Highlights

§ Full-featured electrophysiology package

§ Single program for data acquisition, analysis and hardware control

§ Complex experimental automation

§ Publication-quality graphics

Convenient: All SutterPatch software is run by a single application. No need to launch multiple programs or to move data between programs.

Comprehensive: All data recordings, analyses, graphs, layouts, configurations and controls are saved in a single (daily) experiment file. This ensures that data are always kept together with their complete contexts.

Automation: Automate your experiment using a rich set of data acquisition, data analysis, and (hardware) amplifier controls. Create complex “Paradigms” that can respond to changing conditions via conditional “If – then” steps and “For loops”.

1.3 Experiment Structure

Experiment:

An Experiment is the highest-level structure in the SutterPatch world. An Experiment file (*.pxp) can encompass all SutterPatch activity for the entire day, such as instructions (Paradigms), data acquisition parameters (Routines), recorded data (Series), execution settings, history, and comments. During reanalysis, data can be included from multiple experiments. Typically, one Experiment is created for each cell or preparation recorded from.

Paradigm:

A Paradigm is a sequence of control instructions used in an Experiment. Every Experiment contains at least one Paradigm, whether pre-planned by the user or auto-triggered by the system.

A loaded Paradigm “pool” (file) can contain multiple Paradigms for rapid access and execution. Such “planned” Paradigms can contain simple sequences, or sophisticated control structures, using a rich set of operators, such as conditional processing via “If-then” steps, nested “For loops”, user-defined variables and hardware commands. A planned Paradigm can acquire data by running a Routine.

However, if a Routine is manually run in the Scope window, a “place-holder” auto-triggered Paradigm is created as a default container. This default Paradigm ensures that each Series is associated with a Paradigm in the context of an Experiment. If an auto-triggered Paradigm is already the active Paradigm, it is used for subsequent manually-run Routines.

However, if a Routine is manually run in the Scope window, a “place-holder” auto-triggered default Paradigm is created as a container. This default Paradigm ensures that each Series is associated with a Paradigm in the context of an Experiment. If an auto-triggered Paradigm is already the active Paradigm, it is used for subsequent manually-run Routines.

Figure 1‑1. SutterPatch Data Structure

Top: A planned Experiment with two Paradigms that automatically control Routines.

Bottom: Manual execution of a Routine outside a Paradigm - uses a default “auto-triggered” Paradigm for that Routine.

Paradigm Data:

Paradigm data are a collection of the Paradigm plus all data points, variables, and acquired metadata event tags during the execution of a Paradigm. This allows reconstruction of the exact course of an experiment. While a Paradigm could be compared to an itinerary, the Paradigm Data correspond to the route a journey actually took. If conditional control is used in a Paradigm, e.g., for the number of loop cycles or a decision in an “If-then” step, these actions are recorded in the Paradigm Data, even though they are not predetermined.

Routine:

A Routine is the set of data acquisition and online data analysis parameters that control input and output channel timing, triggering, command waveforms, display and real-time analysis.

A loaded Routine “pool” (file) can contain multiple Routines for rapid access and execution.

Series (Routine Data):

Acquiring data creates a Series composed of all sweeps of data from all input signals. Multiple runs of a Routine create multiple Series of data. All Series are automatically stored in the current Experiment file.

Channel:

A Channel corresponds to a physical input (A/D) or output (D/A) of the IPA system.

Analog input channels are used to record data, and are displayed in their own panes in the Scope window. There are two dedicated internal analog input channels from the IPA headstage (Current1, Voltage1), and four dedicated analog input channels from the Double IPA headstages (Current 1, Current 2, Voltage 1, Voltage 2). Four general-purpose Auxiliary analog input channels (AuxIN1 – AuxIN4) allow recording from external devices via the BNC breakout cable or the optional IPA Patch Panel.

Analog output channels are used to send electrical stimuli, such as analog command waveforms to the preparation. There is one dedicated, internally configured, analog output channel for Stimulus commands to the IPA headstage (StimOUT1), and two dedicated internal analog output channels for Stimulus commands to the Double IPA headstages (StimOUT1, StimOUT2). Two general-purpose Auxiliary analog output channels (AuxOUT1, AuxOUT2) can send output signals via the BNC breakout cable or the optional IPA Patch Panel.

Digital Output bits are also referred to as output “channels”. There are 8 digital output channels (DigOUT1 – DigOUT8) that send output signals via the BNC breakout cable or the optional IPA Patch Panel.

Signal:

Named analog input and output channels are referred to as Signals. A Signal is either the scaled representation of a physical channel, or the virtual result of a computation.

Sweep:

A Sweep is the sum off all data points from all Signals, acquired for a single period from time zero, for a fixed duration. In SutterPatch Software, the Sweep Duration is determined by the duration of the command waveform.

Trace:

A Trace is a Sweep applied to a single Signal. Therefore, a Sweep can be described as the collection of Traces across all Signals.

Segment:

A Segment exists as a user-defined section of the command waveform. Each Segment has a waveform type, an amplitude, and a duration. A command waveform can be composed of up to 50 Segments.

Metadata:

Metadata are additional information associated with stored raw data. These can include information about preparation (cell/tissue/animal), instrumentation (hardware and software), environmental parameters (temperature, atmospheric composition, etc.), stimuli (chemical compounds, light, acoustic, etc.) and other parameters. Metadata information is associated with the running of Paradigms and Routines, and their resulting data.

Metadata are dynamically recorded with a timestamp during an experiment. Information that can be determined by the system, such as the connected hardware, SutterPatch version, user Login Name, or the change of a digital output level, are automatically recorded without user intervention. In addition, the user can enter values for a large number of user-defined Metadata parameters, such as identifiers for the experimental animal or cell, the animal species, age and genotype, information about the recording solutions, and the electrodes or stimuli applied during the experiment. The total number of Metadata parameters SutterPatch can keep track of is ~ 600. User requests for additional parameters can be accommodated.

Terminology Comparison:

A table of equivalent terms to other major electrophysiology software packages:

SutterPatch	Patchmaster	pCLAMP
Experiment	Compound Data	N/A
Paradigm	Protocol	Sequencing Keys
Routine	PGF Sequence	Protocol
Series	Series	Trial
Sweep	Sweep	Sweep
Signal	Signal	Signal
Trace	Trace	Trace
Segment	Segment	Epoch

Table 1‑1: Software Terminology

2. INSTALLATION

2.1 Computer Requirements

Minimum requirements

Operating System: Windows: 7, 8, 10 (64-bit versions)

Most language packs are compatible.

[listed in Control Panel > System]

macOS: 10.11 (El Capitan)

10.12 (Sierra)

10.13 (High Sierra)

[listed in Apple > About this Mac]

CPU (Central Processing Unit): Dual-core

RAM (Random Access Memory): 3 GB

Hard Disk Free Space: 500 MB

Display Resolution: 1024 x 768 (XGA)

Computer Ports: IPA: (1) USB 2.0 High Speed port

Double IPA: (2) USB 2.0 High Speed ports

Note: USB hubs are not supported.

USB 2.0 computer ports are usually implemented with a 'High Speed’ transfer rate, but a slower ‘Full Speed’ specification can sometimes be found on old PC (Windows) computers or USB add-in cards.

To check for High Speed USB 2.0 ports on a PC computer running Windows, look in the Control Panel / Device Manager / Universal Serial Bus controllers section for “Enhanced” host controllers. (USB 3.0 SuperSpeed ports are marked as ‘USB 3.0’.) As this does not provide any mapping information to the computer’s physical ports, and there can be a mix of built-in USB port versions, you should test the physical USB ports for High- Seed performance.

Recommended specifications

RAM (Random Access Memory): 16 GB

Display Resolution: 1920 x 1080 (Full HD)

2.2 SutterPatch System Environment

The SutterPatch software runs in the Igor Pro 7 64-bit system environment. Igor Pro is widely used by scientists to acquire and analyze data, and to create publication-quality presentation graphics.

Igor Pro 7 Features

• High-quality presentation graphics

• High-speed data display

• Large data set handling

• An extensive set of built-in data analyses-

• Waveform arithmetic

• Image display and processing

• Graphical and command-line user interfaces

• Automation

• Extensibility via C and C++ modules

• Extensive online help and PDF manual

The SutterPatch full installer automatically installs Igor Pro 7 (64-bit English version). Igor Pro 7 has a 30-day trial period where it is fully functional and fully supports SutterPatch. After 30-days, if the Igor Pro 7 license has not been activated, Igor Pro 7 runs in a demo mode with limited functionality that does not support the SutterPatch application.

Note: Japanese versions of Igor Pro are not supported by SutterPatch.

For third-party applications that require a 32-bit version of Igor Pro 7:

Windows: The 32-bit version of Igor Pro 7 is included in the installation.

Macintosh: Manually install the 32-bit version of Igor Pro 7:

1. Download the Igor Pro 7 Installer at www.igorpro.net

2. Install Igor Pro 7.

3. Download and install the SutterPatch Updater (https://www.sutter.com/AMPLIFIERS/SutterPatch.html)

2.3 Mounting Instructions

Rack Mounting: The IPA amplifier is ready for mounting in a standard 19” wide equipment rack in a 1U space (DIPA: 2U). A rack mount hardware kit consisting of hex screws, washers and cage nuts is included.

Benchtop Usage: Attach the four included stick-on feet to the bottom of the IPA amplifier.

2.4 Electrical Connections

Typical AC Power: 60 Hz, 120 V

50 Hz, 240V

However, the IPA amplifier will run on power from 88 to 264 VAC. No switches need be set.

The AC power should be as clean as possible:

· At a minimum, a surge protector should be used for high-voltage spikes; if lightning strikes are a concern, it should be rated > 1000 joules and > 40 kA.

· If you experience brownouts or voltage sags, a switching power supply (SPS) can be used to supply clean power to your instruments.

· To protect against power interruptions, use a universal power supply (UPS) for uninterrupted clean power.

Power Switch A power button for the IPA amplifier is located on its front panel. The silver power button lights up blue when the power is ON.

2.5 Install Hardware

Figure 2‑1. Rear of IPA Cabinet

1. Plug the female end of the power cord into the IPA rear-panel power receptacle.

2. Plug the male end of the included power cord into a grounded electrical mains outlet.

Figure 2‑2. Front of IPA Cabinet

3. Set the IPA power button OFF (unlit position).

4. Plug the IPA headstage into the HEADSTAGE port on the front of the IPA amplifier - the amplifier and headstage serial numbers should be the same.

For a Double IPA system, attach the lower serial-numbered headstage to HEADSTAGE 1, and the higher serial-numbered headstage to HEADSTAGE 2, as each headstage is individually tuned for its channel.

WARNING! Hot-swapping of headstages can damage their components and increase noise. Turn off the IPA system power before (un)plugging headstages.

Figure 2‑3. Front of Double IPA Cabinet

5. Plug the IPA I/O “breakout” cable, or the cable to the optional IPA Patch Panel, into the AUXILIARY I/O port on the back of the IPA amplifier.

6. Connect the supplied USB cable to the computer’s appropriate USB port, and to the IPA amplifier’s rear-panel USB port.

7. Connect the included electrode holder(s) to the headstage(s). See the Holder section for holder assembly instructions.

2.6 Install Software

Power on the computer.

A: It is strongly recommended to use the latest SutterPatch software version available.

Download the latest version of SutterPatch installer software from https://www.sutter.com/AMPLIFIERS/SutterPatch.html and choose the “Download” tab. Navigate your file browser to the download file and run it.

B: If internet access is not available, attach the included USB flash drive to your computer USB port, and navigate to the flash drive.

1. Install the software for ‘All Users’ by double-clicking on:

§ Windows: SutterPatch_installer_with_Igor

§ Macintosh: sutterpatch_mac_full.dmg

2. Follow the installer prompts.

· If an existing version of Igor Pro 7 is found, it is recommended to replace it. Make sure to keep a backup copy of all user files and parameters you may have placed into the program folder and its sub-folders.

· If an older version of Igor Pro is found, you can choose to keep it, as different versions of Igor Pro can coexist on the same computer.

3. Upon completion, the installer will report a successful installation.

4. Launch Igor Pro 7 and activate its license as instructed. You will need to enter the Igor Pro 7 Serial Number and Activation Key found in your IPA Quick Start Guide.

5. “Eject” the flash drive, and then unplug it from the computer.

2.7 Test System

2.7.1 Install Model Cell

Attach the model cell to Headstage 1 and tighten the screw collar.

Attach the supplied 1 mm grounding wire to the gold sockets on the headstage and model cell.

If the headstage is not placed in a Faraday cage, completely surround the model cell/headstage assembly with electromagnetic shielding, such as aluminum foil.

Connect the shielding material to the headstage ground wire or connector. A short wire with alligator clips on both ends can be used.

2.7.2 Startup

1. Power-on the IPA amplifier by pressing the silver POWER button on its front – it lights up. (It can take a few seconds for the USB connection to be established.)

2. Launch the Igor Pro (SutterPatch) application by clicking on the ‘Igor Pro 7’ ion.

A SutterPatch splash screen displays while opening files.

Figure 2‑4. Splash Screen

Then the Welcome to SutterPatch “start” window displays.

Figure 2‑5: Welcome Screen

3. Click on the ‘Start’ button in the ‘Welcome to SutterPatch’ window, and the application begins compiling. This process may take several seconds.

4. Specify the experiment file name and storage location when prompted.

5. If the IPA amplifier is OFF or disconnected from the computer, you can choose to reconnect, or start up in hardware emulation mode (IPA or Double IPA amplifier).

Figure 2‑6. USB / Emulation Screen

Note: The emulation mode can only be selected when starting a new Experiment.

6. The Dashboard panel is displayed.

7. Click on the ‘Acquire Data’ icon, and a second level of the Dashboard is displayed.

8. Click on the ‘Control Panel’ icon and the Amplifier Control Panel is displayed.

Figure 2‑7. Amplifier Control Panel

a. If “DEMO” is displayed in the Amplifier Control Panel title bar, you are running in a hardware emulation mode - ensure that the amplifier is “on” and its USB cable is connected, and then run a “New Experiment”.

b. Make sure that the Amplifier Control Panel is in voltage-clamp mode – the VC button at the top of the Amplifier Control Panel should be highlighted in red.

2.7.3 Run a Membrane Test

The Membrane Test is useful for a quick check of the IPA system functionality. It tests the three basic steps necessary for recording in a whole-cell configuration. These test values assume a 5 kHz filter.

1. Go to the Dashboard window and click on the Acquire Data icon.

Figure 2‑8. Dashboard

2. Click on the Membrane Test icon.

Figure 2‑9. Dashboard – Acquisition

3. Set the BATH mode.

This mode simulates placing an electrode into the bath solution and sending a voltage pulse through the solution.

Set the Model Cell switch to ‘Bath’, and click on the Analysis window ‘Bath’ button.

Verify the Pipette Resistance: ~10 MΩ

4. Set the SEAL mode.

This mode simulates an electrode making contact onto a cell and forming a high-resistance gigaohm seal with the membrane.

Set the Model Cell switch to ‘Seal’, and click on the Analysis window ‘Seal’ button.

Verify the Seal Resistance: > 10,000 MΩ

5. Set the CELL mode.

This mode simulates an electrode breaking into a cell and achieving a successful whole-cell patch.

Set the Model Cell switch to ‘Cell’, and click on the Analysis window ‘Cell’ button.

Verify these readings:

Series Resistance: ~10 MΩ

Membrane Resistance: ~500 MΩ

Membrane Capacitance: ~28 pF

6. For a dual-headstage system, move the model cell, ground wire and shielding to Headstage 2, set the Scope window to ‘Headstage 2’, and repeat steps 3 – 5.

3. HARDWARE OPERATION

3.1 IPA Front Panel

The front panel of the IPA system is used for the headstage, external I/O connections and power.

Figure 3‑1: Front of IPA Cabinet

The front panel, from left to right:

HEADSTAGE Connector: For IPA headstage

SCOPE-SIGNAL OUTPUT BNC: A scaled analog output signal of the headstage response signal.

· VC mode: mV/pA Variable gain

(Amplifier Control Panel)

Example: Membrane Test

Model Cell: BATH position (10 MΩ)

Amplitude = 10 mV

Gain = 5 mV/pA

Current response = 10 mV/10 MΩ = 1 nA

(I= V/R)

Scaled output voltage = I * Gain

1 nA * 5 mV/pA

= 5 V

· CC mode: mV/mV Variable gain

(Amplifier Control Panel)

Example: Current Clamp

Gain = 100 mV/mV

Response signal = 10 mV

Scaled output voltage = V * Gain

= 10 mV * 100 mV/mV

= 1 V

SCOPE-COMMAND MONITOR BNC: A scaled analog output of the headstage Stimulus signal (StimOUT).

· VC mode: 10 mV/mV Constant gain

Example: Voltage Clamp

Command voltage: 10 mV

Scaled output voltage: V * Gain

= 10 mV * 10 mV/mV

= 100 mV

· CC mode: 0.5 mV/pA Constant gain

Example: Current Clamp

Command current = 200 pA

Scaled output voltage = I * Gain

= 200 pA * 0.5mV/pA

= 100 mV

COMMAND IN BNC: A scaled analog input that adds an external signal into the headstage Stimulus signal (StimOUT).

• VC mode: 10 mV/mV Constant gain

Example: Voltage Clamp

External Command = 5 mV

Stimulus signal = 20 mV

Total stimulation = (V * Gain) + StimOUT

= (5 mV* 10 mV/mV) + 20 mV

= 70 mV

• CC mode: 200 pA/mV Constant gain

Example: Current Clamp

External Command = 1 mV

Stimulus signal = 5 pA

Total stimulation : (V * Gain) + StimOUT

= (1 mV * 200 pA/mV) + 5 pA

= 205 pA

TRIGGER OUT BNC: Digital Trigger pulse

Automatically sent at the start of continuous acquisition or each triggered sweep (including Membrane Test)

POWER Button: Turn power to unit On/Off. Lights up blue when ‘On’

3.2 Double IPA Front Panel

The front panel of the Double IPA system duplicates the IPA system by adding a second headstage port into a second (upper) row of connectors:

HEADSTAGE 2

SCOPE-SIGNAL OUTPUT

SCOPE-COMMAND MONITOR

COMMAND IN

Figure 3‑2. Front of Double IPA Cabinet

3.3 IPA Rear Panel

The rear panel of the IPA system is used for grounding, operational connections, and I/O. The rear panel of the Double IPA amplifier is essentially the same as for the IPA amplifier.

Figure 3‑3: Rear of IPA Cabinet

< Unlabeled > Power-entry receptacle For AC power cord

TRIGGER IN BNC: Digital sweep trigger (+2.3 V)

AUXILIARY I/O D-Sub 15 connector: Auxiliary analog input and output channels, digital output channels, signal ground.

See Appendix E for pin definitions.

SIGNAL GROUND 4 mm Banana socket: Low-voltage grounding

EARTH GROUND 4 mm Banana socket:

< Unlabeled > USB Type B receptacle: USB 2 computer communications

3.4 Grounding

A connection is needed to the ground outside for an “Earth” ground of your laboratory electrical system. If your building’s electrical grid does not provide an excellent earth ground, sometimes the plumbing system can be used. Or consider creating an earth ground for your own laboratory, using a heavy metal bar penetrating deep into the earth.

The equipment in ~~the~~ a rig should all be grounded to a single point to avoid ground loops. Installing a bus bar to an earth ground can make this easier to accomplish.

~~“Chassis” ground is a protective ground to prevent shocks from the casing of instruments. This carries much less current than an earth ground, but much more than a signal ground.~~

“Signal” ground is ~~the most~~a sensitive ground for very low voltages:

· BNC shields: ~~hard~~Hard-wired to signal ground

· Bath ground electrode: ~~connect~~ Connect to the headstage signal ground jack

· Shielding: ~~connect~~ Connect to the IPA rear panel SIGNAL GROUND socket

However, due to the complexity of grounding factors, despite any preconceived cabling notions, you may need to empirically determine the best grounding configuration of your system. For example, when multiple headstages are used, one or both headstages might need to be grounded.

3.5 Headstage

The IPA headstage supports whole-cell voltage and current clamp in the same headstage. A 1 mm gold pin signal-ground socket is on the back of the headstage.

The length of the headstage cable can be increased with a 6-foot HDMI (non-powered) extension cable.

3.6 Holder

A “holder” attaches a microelectrode (pipette) to a headstage. It provides mechanical stability for the pipette, low-noise for the electrical circuit, and chemical inertness from its physical components. Our pipette holders accept electrode glass in the range of 1.0 – 1.7 mm OD using sized-by-color silicone gaskets.

The standard pipette holder included with the IPA amplifier is composed of low-noise polycarbonate and Teflon. A suction tube projects at a right-angle from the middle of the barrel.

Note: While polycarbonate is a proven material for patch pipette holders, it undergoes significant thermal expansion. Uneven warming can lead to motion of the pipette tip, and is often incorrectly perceived as drift in the micromanipulator. Quartz has a significantly lower thermal expansion coefficient and virtually eliminates thermal drift.

An ultra-low-noise quartz pipette holder is optionally available.

Warning! Quartz is fragile, and can crack or shatter on impact. Treat your quartz pipette holder with the same care as you would with an optical component.

3.6.1 Assembly

The holder is assembled from 8 parts incorporated into a main barrel:

End Cap – Gasket – Silver Wire – Barrel – Tubing – Gold Pin – Pin Cap - Lockdown Ring

Note: The silver wire must be chlorided before use. See “Chloriding Silver Wire”.

Figure 3‑4: Electrode Holder

Figure dimensions are in “inches [ mm ]”.

1. Cut the silver wire to size: the depth it extends into the pipette plus half the length of the barrel.

2. Thread the silver wire through the barrel.

3. Cut a small piece of clear tubing sized to fill the tiny “end-cup” in the pin-side of the barrel – the end with the narrower shaft.

4. Thread the small piece of tubing over the silver wire into the end-cup.

5. Crimp the end of the silver wire just slightly over the end of the tubing.

6. Slide the Lockdown Ring over the tubing-side of the barrel, with the ring’s threads facing outwards.

7. Insert the Gold Pin into the recessed end of the Pin Cap - push it through the pin hole until it stops.

8. Screw the Pin Cap onto the barrel. Pressure from the compressed snippet of tubing ensures electrical contact between the silver wire and the gold pin.

9. Find a silicone gasket with ID (inner diameter) just greater than your pipette OD (outer diameter):

Gasket ID Color

1.1 mm Clear

1.2 mm Green

1.5 mm Orange-Red

1.75 mm Blue

10. Thread the gasket onto the silver wire on the End-Cap side - the side with the narrower shaft.

11. Thread the End Cap onto the silver wire and loosely tighten until it makes contact with the gasket.

12. Carefully thread a solution-filled micropipette onto the silver wire and into the gasket, and then push it into the barrel until it reaches the back end of the bore in the middle of the barrel.

13. Tighten the End Cap onto the barrel.

14. Attach the holder to a headstage with the Lockdown Ring.

Assembly tips

1. Silver wire should be kept straight – do not bend or twist.

2. Ensure good contact between the silver wire and the gold pin.

Check for proper tubing height and wire-crimping length – avoid excess or insufficient amounts.

For the most stable configuration, solder the silver wire to the end of the Gold Pin. Apply only a small bead of solder to the top of the pin in the very middle - avoid any excess solder that might interfere with the parts properly mating, as excess solder can result in air or solution leaks.

3. Fire-polish glass electrodes on both ends to prevent scratching the silver wire or the holder barrel.

4. The rubber gasket will wear out over time and need replacing.

3.6.2 Chloriding Silver Wire

The silver wire should be chlorided before first time use, and then re-chlorinated monthly, or as needed.

Chemical Method

1. If needed, use a razor blade or fine sandpaper to rub off any insulation.

2. Optionally clean the silver (Ag) wire with ETOH (ethanol) to remove finger oils.

3. Immerse the silver wire in common household bleach (sodium hypochlorite) in glassware for 5 – 30 minutes until it turns purple-gray in color.

4. Remove the chlorided silver wire and rinse in distilled water.

5. Dry for storage.

Electrochemical Method

1. If needed, use a razor blade or fine sandpaper to rub off any insulation.

2. Optionally clean the silver (Ag) wire with ETOH (ethanol) to remove finger oils.

3. Connect a silver wire to each pole (positive & negative) of a household battery (1.5 V – 9 V).

4. Immerse the two silver wires in a solution of KCL (3 M) in glassware for 5 – 10 minutes. The wires should not touch each other. Bubbling around the silver wire indicates electroplating is occurring.

Alternatively, use HCL (1M) with a 2 hour immersion time.

5. The charging polarity for the wires should be reversed a few times during the process.

6. A fully chlorided silver wire should be purple-gray in color. Remove the chlorided silver wires and rinse in deionized water.

7. Dry for storage.

Re-Chloriding Silver Wire

1. Pass the used silver wire through a flame - the wire should become bright silver in color.

Alternatively, use a razor blade or fine sandpaper to scrape off any existing chloride.

2. Chloride the wire as described above.

3.6.3 Holder Maintenance

Holders must be properly maintained for good noise performance.

Storage:

1. Holders should be clean and dry.

2. Store in a container with dessicant.

Before 1st time use:

1. Disassemble the holder.

2. Rinse the polycarbonate parts in 70% ethanol.

3. Blot dry.

4. Store in a container with dessicant overnight.

After daily use:

1. Rinse holders with distilled water. For more thorough cleaning, wash with ethanol.

2. Blot dry.

Caution! Washing with soapy water can leave a film.

Continual cleaning with ethanol can degrade the polycarbonate parts.

Do not clean with methanol or strong organic solvents such as acetone.

Weekly Cleaning:

1. At least once per week, disassemble holder.

2. Clean the polycarbonate parts with 10 – 20 s sonication in distilled water.

3. Blot dry.

4. Store in a container with dessicant overnight.

3.7 Maintenance

3.7.1 Inspection

Periodically inspect all cables and connections to make sure that all cables are in good condition, and that all connections are firmly and evenly seated.

3.7.2 Cleaning

Routine cleaning of the IPA system is required to prevent excessive dust accumulations. Wipe all exterior surfaces with a soft dry cotton cloth.

3.8 Calibration

The IPA amplifier can be calibrated in the Set Preferences / Hardware section:

Calibrate Now Trim the unit’s DAC (Digital-to-Analog Converter) to remove its microvolt bias from the output stimuli.

Reset to Zero All calibration settings are zeroed and the holding potential is reset to zero. This is useful in case of problems with the DAC trim levels.

3.9 Amplifier Control Panel (Software)

This software interface controls the IPA amplifier settings. It replaces all physical knobs, dials and meters, such as found on non-computer-controlled amplifiers.

Most of these settings can also be programmatically controlled in a Paradigm.

Figure 3‑5: Amplifier Control Panel

General Controls

1 - 4 Headstage # tabs

When only a single IPA amplifier is being used, headstage selection tabs are not displayed.

When multiple IPA amplifiers or a Double IPA amplifier are used, multiple headstage tabs will display. Each headstage maintains its own settings.

Clicking on a headstage tab will open its last-used active mode (VC, CC) settings. This will also blink the power light of the attached amplifier, which is useful in identifying which headstages are associated with which amplifiers.

Note: If a headstage is unattached while an IPA or Double IPA amplifier is in use, its input channel will be at “ground”. For older IPA amplifiers, the channel might appear as saturated.

V The ‘V’ meter displays the Voltage# input channel reading.

I The ‘I’ meter displays the Current# input channel reading.

VC The ‘VC’ button switches the IPA amplifier into Voltage Clamp mode.

CC The ‘CC’ button switches the IPA amplifier into Current Clamp mode.

The ‘Reset USB’ button displays the state of the USB communication channel between the computer and the IPA system(s).

· A green button indicates that a stable USB connection to the IPA system has been established.

· A red button indicates that there is no USB connection to the IPA system, or that SutterPatch is in Demo (hardware emulation) mode.

· When multiple amplifiers are connected, if any one instrument loses the connection, the button turns red.

· ‘Reset USB’ will reset all USB connections

· It can take several seconds for the USB connection to be re-established

Seal (n) Toggle the ‘Seal’ button “on” to generate a basic seal test pulse directly from the Amplifier Control Panel; monitor the Current signal to observe the response.

The Seal button number is the amplitude of the test pulse.

The low/high state of the pulse amplitude is displayed beneath the button by a slider control. Switch between states by clicking on or by dragging in the slider control.

· VC Mode: The low/high values are in mV.

· CC Mode: The low/high values are multiplied by 20 pA.

The pulse amplitudes are set in the Amplifier Control Panel right-click menu ‘Set Seal Test’.

View the Seal Test signal by connecting the IPA amplifier ‘SCOPE’ BNCs to an oscilloscope. Numeric responses are also displayed in the Amplifier Control Panel “V” and “I” meters.

Note: The hardware Seal Test in the Amplifier Control Panel uses and maintains different hardware settings than the Membrane Test.

Right-click menu Click almost anywhere in the Amplifier Control Panel to access:

· Reset USB Re-establish the USB connection to the IPA amplifier. If you are in demo mode and a powered IPA is attached, demo mode is disabled and the program runs “live” with the hardware.

· Set Seal Test Configure the IPA amplifier seal test pulse amplitudes.

Enter low seal test amplitude: [ ±100 ]

Enter high seal test amplitude: [ ±100 ]

· VC mode: Amplitude unit is mV.

· CC mode: Values are multiplied by 20 pA.

Note: The ‘low’ amplitude value can be set greater than the ‘high’ amplitude for negative polarity test pulses.

· Reset Amplifier Controls Reset the hardware settings of an attached Sutter amplifier to their factory defaults, which also resets the Amplifier Control Panel values.

Offset The amount of offset applied is displayed in the numeric field below the Offset button

[ ±250.000 mV ]

The electrode offset should be initially adjusted in voltage-clamp mode, when the electrode is placed into the bath solution. This adjusts the electrode current signal to zero, providing an accurate reference baseline for measurements. This offset counteracts any inherent hardware circuitry offsets, as well as electrode-in-the-solution offsets.

Click on the Offset button to apply an offset that zeroes the signal. The “auto-offset” value is an approximation that might need further adjustment.

Note: In Demo mode, this value is set to -0.200 mV.

Numeric values can be directly typed into the numeric field, or

· For fine adjustments, use the numeric field up and down spinners to increase or decrease the setting by ~0.015 mV.

Note: The Offset spinner step size (~0.015 mV) is based on the 16-bit resolution of a 1 V DAC. The actual spinner step size resolution is 0.0152588 mV.

· For moderate adjustments, increase the spinner increment by 10x (~0.15 mV) by holding down the Shift key and simultaneously clicking on the spinners.

· For fastest operation, select the offset field, hover over the numeric field or spinners, and hold down the Shift key while simultaneously scrolling up or down with the mouse wheel.

Lock Once an electrode offset has been applied, use the ‘Lock’ check box to prevent accidental changes to the Offset.

Voltage Clamp Controls

VC The ‘VC’ tab displays amplifier Voltage Clamp controls:

Holding (mV): [ ±1000 ]

After achieving a seal, the holding voltage is typically set to the cell’s equilibrium or “resting” membrane potential (typically -60 to -80 mV for neurons).

Whenever you enable or leave the Amplifier Control Panel, a tag is written to the Log window with the Holding changes.

Electrode Compensation: Electrode capacitance compensation

Mag (pF): [ 0.00 – 25.00] Magnitude

Phase (%): [ 0 – 100 ]

Auto Automatically sets approximate values

Compensate for the microelectrode capacitive transients by clicking the ‘Auto’ button, and then iteratively adjusting the ‘Mag’ and ‘Phase’ controls. For a square pulse (Seal Test) command, the goal is to eliminate the sharp (fast) transients and reproduce a square waveform with minimal distortion.

Cell Compensation Whole-cell capacitance compensation

Rs (MΩ): [ 0 – 100.0 ] Series Resistance

Cm (pF): [ 0 – 100.0 ] Membrane Capacitance

Auto Automatically sets approximate values (uses small “gentle” steps to avoid hyperpolarization)

After breaking into a cell, i.e., going “whole cell”, capacitive transients are now generated by the entire membrane of the cell, also producing a significant slow decay phase in the signal.

To remove the transients, click ‘Auto’, then iteratively adjust the ‘Rs’ and ‘Cm’ controls until the signal is adequately compensated (smooth transitions without transients.)

Note: When the Membrane Test is running, ‘Cell Compensation’ must be disabled, else capacitance spikes are suppressed, and Series Resistance and Membrane Capacitance values cannot be calculated.

The IPA system is optimized for real-world measurements from electrodes. But when used with the model cell, the compensation might need several more ‘Auto’ adjustments to compensate the model cell capacitance.

Rs Correction Series resistance compensation

“Rs Correction” speeds up the rise time of the current response, reduces unwanted filtering effects, and corrects voltage drops.

Corr (%): [ 0 – 100 ] Correction

Pred (%): [ 0 – 99 ] Prediction

Lag (µs): [ 20 – 200 ] (RC filter component)

Lag = 1 / (2 * π * Bandwidth)

Rs Correction first requires the Electrode and Cell Compensations to be applied.

Then set the Prediction (Pred) to “supercharge” the command potential. Small transients should become visible at the beginning and end of the current response.

Next, increase the Correction (Corr) current injected into the membrane. As the Corr setting is increased to sharpen the rise time, the current response transients increase in size. Try to avoid overshooting - if the correction is set too high, internal feedback can lead to oscillation of the circuit, i.e. “ringing”, and loss of a patch. Reduce oscillation of the circuit by adjusting the ‘Lag’ setting - larger values increase the stability of the circuit, but also increase the rise time.

Remove the Prediction/Correction transients in the signal by reducing the Cell Compensation ‘Rs’ setting until a minimum value is found.

Finally, adjust the Cell Compensation ‘Rs’ setting again until the best result is achieved, or try over again with lower Prediction/Correction settings.

Gain Input Gain

mV/pA Signal Range

0.5 ± 20 nA

1 ± 10 nA

2.5 ± 4 nA

5 ± 2 nA

10 ± 1 nA

25 ± 400 pA

Filter Low-pass 4-pole Bessel filter.

kHz

0.5

Filter settings for the active IPA headstage primary analog input signal (VC mode: Current, CC mode: Voltage) are applied when the Scope window is created.

Note: Demo mode bypasses the filter.

Current Clamp Controls

CC The ‘CC’ tab displays the IPA Current Clamp controls:

H. Current (pA): [ ±1000 ]

A Holding Current can be preset here. Use the Enable checkbox to activate it.

Electrode Compensation

Mag (pF): [ 0.00 – 25.00 ] Magnitude

Phase (%): [ 0 – 100 ]

After seal formation (i.e., in a cell-attached patch configuration), compensate for the microelectrode capacitive transients by iteratively adjusting the controls. For a square pulse (or Seal Test) command, the goal is to eliminate the sharp (fast) transients and produce a square waveform without distortion.

Bridge Balance

Resistance (MΩ): [ 0 – 200.0 ]

Bridge balance compensation is used to correct distortion of the signal due to a voltage drop across the electrode during current flow.

Tracking

Potential (mV): [ ±1000 ]

Slow tracking is used to keep the membrane voltage at a set target level without drifting over time. Enter the desired cell potential to be maintained.

The Tracking time constant (tau) is ~18 ms, equating to a rise time of 40 ms, or a bandwidth of 8.75 Hz.

Note: When Tracking is enabled, the Holding Current is automatically disabled, as the current output is dynamically adjusted by the system.

Gain

Input Gain

mV/mV Signal Range

10 ±1 V

20 ± 500 mV

50 ± 200 mV

100 ± 100 mV

200 ± 50 mV

500 ± 20 mV

Gain settings for the active IPA headstage are applied to its primary analog input signal (‘Voltage’ in CC Mode) when the Scope window is created.

Filter Low-pass Filter

kHz

0.5

Filter settings for the active IPA headstage are applied to its primary analog input signal (‘Voltage’ in CC Mode) when the Scope window is created.

Note: Demo mode bypasses the filter.

I/O Controls

I/O The ‘I/O’ tab contains the Digital Output and Auxiliary I/O controls. For a Double IPA system, the I/O tab is only visible when Headstage 1 is selected.

Digital Output

This section controls the holding bit pattern generated by the Digital Outputs of the IPA amplifier. Eight TTL-compatible digital channels are displayed. Switch between digital states by clicking on a channel slider ‘high’ or ‘low’ end, or by clicking and dragging the slider button. When a channel state changes, its dot also changes color:

· high: Red dot “On” (+3.3 V)

· low: Black dot “Off” (0 V)

Note: When multiple Sutter amplifiers are connected, the Digital Outputs are only active for the main amplifier.

Auxiliary I/O

General purpose “auxiliary” analog input/output channels are available.

AuxOUT 1 & 2: [ ±10 V ]

Select an auxiliary analog output channel from the drop-down list. Directly edit its voltage level, or use the spinners to change the value in 0.100 V increments.

When switching between auxiliary channels, hardware output channels maintain separate values, while demo output channels are reset to zero.

Tip: When the IPA system is used as a data acquisition system for external instruments, the auxiliary outputs can be used as holding levels.

Set Click the ‘Set’ button to apply the value to the selected output channel.

AuxIN 1 – 4: [ ±10 V ] (Read Only)

Select an auxiliary analog input channel from the drop-down list.

Read Click the ‘Read’ button to display a single-point reading of the selected input channel.

Tip: This is useful for monitoring slow-changing parameters, such as temperature.

The vertical Show/Hide button displays/hides the VC-CC-I/O panes.

The horizontal Show/Hide button displays/hides the Double IPA headstage monitor, which displays the Voltage and Current values for both headstages.

Figure 3‑6. Headstage Monitor

3.10 Using Peripheral Equipment

The IPA system can control peripheral equipment, such as:

· solution changers

· light sources

· wavelength switchers

· pulse generators

Auxiliary analog output signals can be used to control other instruments within a range of ±10 V. Digital outputs use TTL-compatible voltage signals. Analog and digital holding levels are set in the Amplifier Control Panel

The digital command output can be formatted as either an 8-bit “word” or a single “bit”, as selected in the Routine Editor / Output Channels & Waveform section. Actual command output patterns are configured in the Routine Editor / Output Channels & Waveform / Waveform Editor.

Note: The Analog and Digital controls in the Amplifier Control Panel provide a way to quickly and easily test the behavior and operation of peripherals, without the need to create or modify Routines.

3.11 Using Multiple Sutter Amplifiers

A combination of any two IPA or Double IPA amplifiers can be connected and run simultaneously by the SutterPatch program, providing up to 16 input channels. However, Digital Outputs are only available for the main Sutter amplifier.

The “main” amplifier controls the timing for the “secondary” amplifier. Connect the main amplifier front panel TRIGGER OUT BNC to the secondary amplifier rear panel TRIGGER IN BNC to provide the clock signal between the two amplifiers.

Tip: When the amplifiers are powered up, the computer USB ports are “enumerated” and receive a sequence number. This sequence should not change unless the computer is rebooted, or the USB-attached equipment is changed, or the USB ports are re-configured. The Amplifier Control Panel displays each connected Sutter amplifier headstage using a numbered tab for each headstage – you will need to test for the headstage sequence on power-up.

Note: Emulation (demo) mode is not supported for multiple-amplifier configurations.

3.12 Using Non-Sutter Amplifiers

The IPA system can also be operated as a stand-alone data acquisition system interfacing to non-Sutter amplifiers.

BNC connectors are located on the IPA rear panel Auxiliary I/O cable or optional Patch Panel.

AuxOUT1 & 2 These two auxiliary analog output channels can be used to send stimulus waveforms to external instruments, such as non-Sutter microelectrode amplifiers.

AuxIN1 – 4 These four auxiliary analog input channels can be used to digitize signals from external instruments, such as non-Sutter microelectrode amplifiers.

DigOUT1 – 8 Digital output patterns can be sent via eight digital output channels to a variety of peripheral equipment.

Auxiliary analog and digital holding levels are set in the Amplifier Control Panel I/O tab.

Note: Sutter amplifier output levels into Sutter systems attenuate by < 0.2%.

HEKA amplifier output levels into Sutter systems attenuate by 0.%.

Axon amplifier output levels into Sutter systems attenuate by 5%.

3.13 Using Non-Sutter Data Acquisition Systems

The IPA system can also be operated as a stand-alone amplifier using non-Sutter data acquisition systems, while the IPA amplifier continues to be controlled via the Amplifier Control Panel.

IPA System Front Panel Connections

BNC Channel

COMMAND IN Stimulus to the preparation

Combines the analog input signal from an external source with the stimulus (command waveform) sent to the preparation.

The Command In external signal is summed with the IPA internal StimOUT output signal, and is then sent to the IPA headstage.

SCOPE - SIGNAL OUTPUT Data from the preparation

This BNC supplies the response from the IPA preparation.

The current or voltage response from the headstage is directly available from this analog BNC output, and can be connected to an external data acquisition system for digitization and recording.

SCOPE - COMMAND MONITOR Data from the Stimulus signal

This BNC allows you to monitor the stimulus channel.

The analog stimulus delivered to the IPA headstage (voltage or current) is directly available on this BNC, and can be connected to an external data acquisition system for digitization and recording

4. SOFTWARE OPERATION

4.1 Acquisition

SutterPatch acquisition operations.

4.1.1 Camera Module

The SP_Camera window displays:

Camera Select a camera name from those attached to the computer.

Start View live video

Capture Take a single picture. If live video is running, this will take a picture while live video continues to run. An image time-stamp is entered into the Log window.

[video screen]

Last Capture A thumbnail of the last picture taken in the Experiment is displayed.

All pictures are stored in the current Experiment. To review pictures, go to:

Data Navigator Select a Paradigm or Routine. Any associated images are listed in the Preview pane. Click on an image name to display the image.

Data Browser Navigate to the Data / Images folder. Right-click on an image name, and select ‘New Image’.

Tip: For dark-room experiments, the window background color can be adjusted by the operating system.

Windows: In the Control Panel / Appearance / Personalization window, scroll down and select the High Contrast Black theme, or use the Magnifier tool with option ‘Turn on color inversion’ enabled.

macOS: Press ‘Control-Option-Command-8’ to set the System Preferences / Accessibility / Display / Invert Display colors option, or open its menu with ‘Command-Option-5’.

Full-Camera drivers have been successfully tested for the following camera models:

Sentech drivers:

STC-MC33USBVGA

STC-MCS231U3V

STC-MB83USBVGA

STC-MBCM401U3V

STC-MBCM401U3V-NIR

STC-HD203DV

Photometrics PVCAM drivers :

Photometrics: Prime 95B

Prime 95B 25mm

Qimaging: Electro

4.1.2 Free Run

The Free Run (Scope) window simulates a one-channel oscilloscope, and is a quick method of viewing repetitive data. It operates similarly to the Scope acquisition window, with unsupported controls removed or disabled.

After selecting this operation, an Input Channel dialog needs to be configured:

Select channel: Select an input channel from a list of all input channels.

The default channel is ‘Current1’ in VC mode, and ‘Voltage1’ in CC mode.

Select sampling frequency: 1, 2, 5, 10, 20, 50 kHz

Input sweep length (s):

4.1.3 Membrane Test

The Membrane Test is primarily used to monitor seal formation and cell health in a voltage-clamp whole-cell patch-clamp configuration. Its Scope window controls operate similarly to the Scope acquisition window.

Scope Window

The default Scope window top pane displays the Current signal from the active IPA headstage, and the bottom pane displays the corresponding Voltage command signal (a square pulse). For a Double IPA system, switch between headstages by selecting the headstage number in the Amplifier Control Panel.

Membrane Test timing parameters are set in the Preferences dialog:

· Sampling rate: 20 kHz / 50 kHz

· Duration: 20 ms / 200 ms

Note: The 20 ms sweep duration repeats every 100 ms.

The Membrane Test amplitude is set in its ‘Analysis’ sub-window:

· Pulse Amplitude: [ ± 100 mV ]

(Relative to the ‘Holding’ level in the Amplifier Control Panel.)

Analysis Window

The Membrane Test Analysis window contains the three basic steps to form a whole-cell seal:

1) Bath With a new pipette in the bath solution, a low-resistance square pulse is visible. The pipette resistance should be very low if the tip is not clogged.

With dissociated cells, typical pipette resistances are 1 – 5 MΩ. In slice patch experiments, pipette resistances well above 10 MΩ are not uncommon.

2) Seal When an on-cell patch is formed between the pipette and the cell, voltage transition spikes are visible. The seal resistance increases as the seal forms. The goal is to achieve a “gigaseal” with a resistance above 1 GΩ.

3) Cell After breaking through the cell membrane and a whole-cell patch is created, membrane resistance and capacitance measures are calculated from the resulting capacitance spikes. These values can be periodically checked to monitor the health of the cell.

The Membrane Test calculations are displayed in real-time numeric fields:

Bath Pipette Resistance (MΩ) (Model cell = ~10 MΩ)

Seal Seal Resistance (MΩ) (Model cell > 10,000 MΩ

Cell Series Resistance (MΩ) (Model cell = ~10 MΩ)

Membrane Resistance (MΩ) (Model cell = ~500 MΩ)

Membrane Capacitance (pF) (Model cell = ~28 pF)

Cell mode requires that ‘Cell Compensation’ in the Amplifier Control Panel be disabled, otherwise capacitance spikes are suppressed, and Membrane Capacitance and Series Resistance cannot be calculated - a ‘Cell Compensation ON’ notice displays.

Note: ‘Series Resistance’ and ‘Access Resistance’ are equivalent terms.

Write to Log Click the ‘Write to Log’ button to copy the measurements to the Log window.

Note: IPA measurements are notated with “(HS#1)”; Double IPA measurements are notated with the active headstage, “(HS#1)” or “(HS#2)”.

Amplitude Set the amplitude of the square wave pulse [ ±0.100 V ]

Zap [ 0.1 – 2.0 ms ] [ 0.1 – 1.0 V ] (duration, amplitude)

After a gigaohm patch has been achieved, use Zap in the Seal mode as an alternative to suction in creating a whole-cell patch.

Click the Zap button to send a single square wave voltage pulse from the IPA headstage to the preparation to disrupt the cell membrane.

4.1.4 Paradigm Editor

The Paradigm Editor is an advanced feature that opens up a world of complex experimental control via Paradigms and Paradigm Pools. A rich set of operators and actions are available to control and/or automate data acquisition and analysis.

The Paradigm Editor allows you to create “Planned Paradigms”, which offer almost unlimited flexibility in creating and/or automating your patch-clamp experiments, such as running Routines and directly controlling amplifier settings.

Figure 4‑1. Paradigm Editor

Loaded Paradigms display on the left, while loaded Paradigm Steps display on the right. A bottom section can display interactive checkbox controls and/or variables.

Controls

Start Paradigm Run the selected Paradigm highlighted in the Paradigm Pool list. A “Planned Paradigm” is created, that terminates after the end of the last paradigm step.

This button turns into a ‘Stop Paradigm’ button when the Paradigm is running.

Pause Temporarily halt a running Paradigm.

Stop Acquisition End a Routine’s acquisition.

Stop at End of Sweep Wait until the sweep-in-progress has completed before stopping an acquisition.

Timer A running clock displays the time in “hh:mm:ss” since the last timer reset, or since a new experiment established a USB connection or emulation mode.

Reset: Reset the Timer to 00:00:00.

Current Paradigm: The name of the currently loaded Paradigm.

Step: The highlighted Paradigm Step.

Paradigm Status: Status information about Paradigm execution.

Set Tag A comment tag can be manually triggered with this button. Enter the optional tag text in its field. A drop-down list maintains the most recently used entries. Click this button to activate the tag text. The last selection is used for all subsequent recordings within the same Paradigm.

When this button is clicked while acquisition is not running, metadata ‘Tag Comment’ entries are created during acquisition without time stamps.

When this button is clicked during acquisition, a black vertical “tag” cursor is displayed in the Scope window data at that time point, and time-stamped metadata entries are created.

Note: In emulation mode, tag timing is not accurate, and no tags are set in the first sweep.

Tip: When using the Set Tag button, you can reduce the footprint of the Paradigm window by hiding its Editor Controls and Variables,

Show/Hide Editor Controls The Paradigm Editor controls (and checkboxes) for the Paradigm Pool and Paradigm Steps can be displayed or hidden.

Show/Hide User Checkboxes Checkbox controls are displayed at the bottom of the Paradigm Editor controls, for use in conditional Paradigm step execution. This display is dependent upon Show Editor Controls.

Show/Hide Variables: A Variables table can be displayed at the bottom of the Paradigm Editor. These paradigm variables can be utilized in any equation.

Variable names can be edited to any label, but they are only informational, and are not supported in equations.

Last key: The last key (or key combination) pressed on the keyboard is displayed here, such as used in Shortcuts or the ‘If’’ and ‘ElseIf’’ Paradigm steps. (See sample Paradigm ‘Tuning with Keys’.)

Note: Function and Control (Ctrl/Cmd) Shortcut key combinations are not displayed.

Paradigm Pool Files These operations affect the entire “Paradigm Pool”.

New Paradigm Pool Create a new blank Paradigm Pool and optionally copy Paradigms into it from the existing Paradigm Pool.

The suggested name is auto-incremented from the previously loaded Paradigm Pool name.

Load Paradigm Pool Load the Paradigms of a previously saved Paradigm Pool file into the Paradigm Pool.

Revert to Last Saved Undo any unsaved changes to the Paradigm Pool.

Save Paradigm Pool Save the Paradigm Pool using its existing file name and path.

Save Paradigm Pool As Save the Paradigm Pool to a new file, and switch to the new file. The default file name is the same as the original file name.

Save Paradigm Pool Copy Save the Paradigm Pool to a new file, but do not switch to the new file. The default file name has ‘Copy of’ prepended to it.

Merge Paradigm Pools Insert the Paradigms from a previously saved Paradigm Pool file into the loaded Paradigm Pool.

[ The file path and file name of the loaded Paradigm Pool file are displayed.]

Paradigm description: A user description of the active paradigm.

Paradigm Pool A column of paradigm names from the loaded Paradigm Pool.

· Click on a paradigm name to highlight it as the active paradigm and display its steps.

· Double-click on a paradigm name to start execution of the paradigm and display its steps.

· Click-and-drag a paradigm name to change its position in the column.

Save Pool Save the Paradigm Pool using its existing file name.

New Paradigm Create a new blank Paradigm in the Paradigm Pool.

Rename Rename the selected Paradigm.

· Valid characters are A-Z, a-z, 0-9, and “_”.

· Special characters are not allowed.

· Spaces are replaced by an underscore.

· The name cannot start with a number – such entries will have an ‘X’ prepended to the name.

Duplicate Add a copy of the selected Paradigm to the Paradigm Pool. The Paradigm name number is appended or incremented.

Delete Remove the selected paradigm from the Paradigm Pool.

Revert Select a paradigm and click the ‘Revert’ button. All editable steps are reset to their originally loaded values, as long as the Paradigm Pool has not been saved.

Paradigm Steps A column of instructions from the active paradigm is displayed. These instructions are sequentially run by the paradigm.

· Click on a paradigm step to highlight it as the active step.

· Double-click on a paradigm step to view or edit its settings.

· Click-and-drag a paradigm step to change its position in the column.

Note: Step values are usually in standard units, i.e. "Volts" and "Amperes".

Step Buttons

Insert Insert a new command Step into the Paradigm Steps column:

Amplifier

Each Sweep

Routine

------------

Analysis

Camera

Clear Key

Execute

Export

Front Window

Hide Window

Reset Timer

Scope Operation

Set Axis

Set Checkbox

Set Solution

Set Tag

Set Variable

Sound

View Last

Write Log

------------

Alert

Beep

Comment

Wait

------------

Break

Chain

For Loop

Jump

Label

------------

ElseIf

Else

Edit If a highlighted Step is configurable, clicking the Edit button (or double-clicking the step) will open it in the Paradigm Steps Editor for configuration.

Also, if a highlighted Step’s text is partially hidden, use the Edit button to view the entire entry.

Toggle Skip Mark a step so it is not executed.

A semicolon is prepended to the Step number to “comment out” the instructions, and a Skip status is appended to the Step text.

Example: A ‘Beep’ command in Paradigm step #2:

; 2 Beep, Skip=true

The leading semicolon ";" prevents this step from being executed by the instruction queue, and the ‘Skip’ status is displayed.

Duplicate Insert a copy of the selected step.

Revert Select a Step to be reverted, and click the Revert button. Editable fields are reset to their originally loaded values, as long as another Paradigm has not been loaded.

Delete Delete the selected step. For multi-line steps, optionally delete the entire group.

Step Execute the selected step, then move to the next step.

Executing a single step does not terminate a running Paradigm, even if it is the last step in the Paradigm.

Note: A ‘For’ loop is processed as a single step.

Step to End Execute the selected step and all following steps as fast as the system allows

Slow Speed Execute ‘Step to End’ at ~1 second per step.

Log Main Steps Action-oriented steps are recorded in the Paradigm metadata (visible in the Metadata ‘By Event’ view):

Amplifier

Break

Camera

Chain

Execute

For Each Sweep

Reset Timer

Routine

Set Checkbox

Set Solution

Set Variable

Wait

Log All Steps Log the main steps and additional steps into the Paradigm metadata (visible in the Metadata ‘By Event’ view.)

Insertable Steps…

Amplifier

Control the IPA amplifier hardware.

Figure 4‑2. Step: Amplifier

Default Setting: Amplifier, Target=VHold, Equation=-0.080

Undo Removes any unsaved edits to this step.

[ drop-down list ] Amplifier settings:

SelectProbe (select active probe)

Most amplifier commands target the “active” probe - the headstage presently controlled by the Amplifier Control Panel. When multiple Sutter headstages are attached, you can change which probe will be the target of the next amplifier command.

CCMode (amplifier current clamp)

VCMode (amplifier voltage clamp)

Hold (IHold in CC-mode, VHold in VC-mode)

[ ±0.000,000,020 A (±20 nA), or ±1.000 V (±1000 mV) ]

IHold (amplifer holding current, A)

[ ± 0.000,000,020 A (±20 nA) ]

IHoldOn (amplifier holding current On)

VHold (amplifier holding voltage, V)

[ ±1.000 V (±1000 mV) ]

VHoldOn (amplifier holding voltage On)

IGain (amplifier current gain, V/A)

Set the gain for the active ‘Current’ input channel using standard unit numbers (V/A) or scientific notation (1 mV/pA = “1e9”.). The value is converted to a preset Gain setting:

· 0.5 mV/pA

· 1 mV/pA

· 2.5 mV/pA

· 5 mV/pA

· 10 mV/pA

· 25 mV/pA

To help reduce signal saturation from too high a gain, a 90% threshold promotes the equation value to the next higher Gain setting.

VGain (amplifier voltage gain, V/V)

Set the gain for the active ‘Voltage’ input channel using standard unit numbers (V) or scientific notation (1 mV = “1e3”). The value is converted to a preset Gain setting:

· 10 mV/mV

· 20 mV/mV

· 50 mV/mV

· 100 mV/mV

· 200 mV/mV

· 500 mV/mV

To help reduce signal saturation from too high a gain, a 90% threshold promotes the equation value to the next higher Gain setting.

Filter (amplifier input filter, Hz)

Apply a preset filter level to the input channels:

· 500 (500 Hz)

· 1000 (1 kHz)

· 2000 (2 kHz)

· 5000 (5 kHz)

· 10000 (10 kHz)

· 20000 (20 kHz)

To help prevent over filtering, a 10% threshold promotes the equation value to the next higher filter level.

Offset (amplifier pipette offset, V)

[ ±0.5 (±500 mV) ]

OffsetLock (amplifier pipette offset lock On)

[ 1 = On, 0 = Off ]

VTrack (amplifier tracking potential, V)

[ ±1 (±1000 mV) ]

Set a value and enable slow CC Tracking

VTrackOn (amplifier tracking potential On)

[ 1 = On, 0 = Off ]

Enable slow CC Tracking

ECompMag (amplifier electrode compensation magnitude, F)

ECompPhase (amplifier electrode compensation phase, fraction)

ECompOn (amplifier electrode compensation phase On)

[ 1 = On, 0 = Off ]

CmComp (amplifier cell compensation Cm, F)

Set a cell capacitance value and enable cell capacitance compensation

RsComp (amplifier cell compensation Rs, Ohm)

Set a series resistance value and enable cell capacitance compensation

RsCompOn (amplifier cell compensation On)

[ 1 = On, 0 = Off ]

RsLag (amplifier Rs correction lag, s)

RsPred (amplifier Rs prediction, fraction)

[ 0.00 – 1.00 ] Converted to a percentile

RsCorr (amplifier Rs correction, fraction)

[ 0.00 – 1.00 ] Converted to a percentile

RsCorrOn (amplifier Rs correction On)

[ 1 = On, 0 = Off ]

Bridge (amplifier bridge balance, Ohm)

Reset (reset amplifier controls)

AutoEComp (amplifier auto electrode compensation)

AutoCellComp (amplifier auto cell compensation)

AuxOUT1 (Auxiliary Output-1, V)

AuxOUT2 (Auxiliary Output-2, V)

DigOUTWord (Digital Output Word)

DigOUT1 (Digital Output-1)

DigOUT2 (Digital Output-2)

DigOUT3 (Digital Output-3)

DigOUT4 (Digital Output-4)

DigOUT5 (Digital Output-5)

DigOUT6 (Digital Output-6)

DigOUT7 (Digital Output-7)

DigOUT8 (Digital Output-8)

[ Equation field ] A free-form text field. This field is evaluated and its value passed to the “target” function.

[ Errors are reported under this field. ]

Note: Values in the Equation field are rounded to whole numbers.

While Amplifier steps are configured in standard units (Amperes, Volts), the Amplifier Control Panel displays values in scaled units.

Check Equation Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports “Syntax is ok.”

Insert special identifier Special functions for use in equations. (See list below.)

Each Sweep

Control the Paradigm operations on a “per sweep” basis of a Routine.

Commands to be executed are inserted between the “EachSweep, Target” line and the “ForEachEnd” line.

Figure 4‑3. Step: Each Sweep

Default Setting: ForEachSweep

EachSweep, Target=untitled

ForEachEnd

Undo Removes any unsaved edits to this step.

[ drop-down list ] Select a Routine name from the loaded Routine Pool.

For example, when tracking whole-cell Rs values in real-time measurement graphs, update the Cell Compensation sweep-by-sweep, by inserting an Amplifier step (with its AutoCellComp command selected) within an EachSweep loop

Note: When using ‘Each Sweep’ to record data, the minimum sweep start-to-start time is +200 ms. For faster execution times, use the ‘Routine’ step.

Routine

Execute a Routine and record a data Series.

Figure 4‑4. Step: Routine

Default Setting: Routine, Target=untitled

Undo Removes any unsaved edits to this step.

[ drop-down list ] Select a Routine name from the loaded Routine Pool.

Note: The time from starting this command to recording data is +300 ms.

“Single-stepping” this command (when no Paradigm is running) will create an auto-triggered Paradigm.

Analysis

Save an analysis to the Analysis Editor, or combine it with prior analyses.

Figure 4‑5. Step: Analysis

Default Setting: Analysis, Operation=Save to Editor

Undo Removes any unsaved edits to this step.

Operation

· Save to Editor Save the latest analysis

· Append to Last Append to the prior analysis

· Average with Last Average with the prior analysis

· Show Table Display analysis as numeric table

· Show Graph (1 – 8) Display analysis as visual graph

Camera

Take a single picture and/or run a live video preview. A Camera window is opened behind the Paradigm Editor and Scope windows.

Figure 4‑6. Step: Camera

Default Setting: Camera, Camera=_Camera_Name, Capture =true

Undo Removes any unsaved edits to this step.

Camera Select a camera on the computer system.

Capture Take a picture when executed.

Live view: Configure the state of the live view:

· No Change Keep last settings

· Stop Stop live view

· Start Start live view

Clear Key

Clears the ‘Last key’ field of the last used Shortcut Action keys.

Default Setting: ClearKey

Execute

Extend the functionality of SutterPatch by running an Igor command.

Figure 4‑7. Step: Execute

Default Setting: Execute, Command=Beep

Undo Removes any unsaved edits to this step.

Command Run any Igor command accepted by the Command window, including user-created Functions.

Two special identifiers can also be used:

· t[#] n’th input trace

The trace wave of the channel in Scope Position "n" is accessed - this substitutes for the data folder path.

· p[#] n’th Paradigm variable

Note: Igor syntax usually requires that open/close parentheses “( )” be appended to the end of a command, however exceptions include the “beep” and “print” commands for which no parentheses are used.

Example 1: Reset the Timer - the command would be:

Paradigm_ResetTimer()

Note the open and close parentheses at the end.

Example 2: Create a FFT graph of your data:

1. ForEachSweep

2. EachSweep, Target=YourRoutineName

3. Execute, Command=FFT/OUT=3/DEST=Voltage1_FFT t[2]

4. If, Left=sweep, Operation=”=”, Right=1

5. Execute, Command=Display Voltage1_FFT

6. EndIf

7. Execute, Command=SetAxis Bottom 0,60

8. ResetTimer

9. ForEachEnd

In Step 2: Replace “YourRoutineName” with your own Routine name, or use the sample “IV” Routine.

In Step 3: The Igor ‘FFT’ command is run, and “t[2]” retrieves the Scope’s second input trace.

Export

Export data graphs into a Layout window.

Figure 4‑8. Step: Export

Default Setting: Export, Signal=Layout

Undo Removes any unsaved edits to this step.

[ drop-down list ]

Append Append graphs to an open Layout window, or into a new window.

Create Create a new Layout window.

Graphs per page Set the graph layout configuration for new Layout windows:

1 Graph fills entire page

2 Graphs stacked

3 Graphs stacked

2 x 2 matrix

2 x 4 matrix

[ drop-down list ] Select signals to be exported from a list of default names.

Clear Clear the signal field, set it to ‘off’.

All Selects all entries.

All Signals Selects all input signals.

< List of input signals >

All Analyses Selects all Analysis graphs

< List of Analysis graphs >

[ List of selected signals] User-edited names can be directly entered into the signal field.

Note: The sequence of signals is not used for positioning in the Layout window – signal positioning is based on their Scope window sequence.

Front Window

Set the specified window as the front window.

Figure 4‑9. Step: Front Window

Default Setting: Front Window, Target=Scope Window

Undo Removes any unsaved edits to this step.

Front Window Analysis Editor

Camera Window

Control Panel

Dashboard

Data Navigator

Equation Editor

Log Window

Paradigm Editor

Routine Editor

Scope Window

Shortcut Editor

Solution Editor

Template Editor

Hide Window

Hide the specified window.

Figure 4‑10. Step: Hide Window

Default Setting: HideWindow,Target=Scope Window

Undo Removes any unsaved edits to this step.

Hide Window Analysis Editor

Camera Window

Control Panel

Dashboard

Data Navigator

Equation Editor

Log Window

Paradigm Editor

Routine Editor

Scope Window

Shortcut Editor

Solution Editor

Template Editor

Reset Timer

Reset the Paradigm Editor Timer to 00:00:00.

Default Setting: ResetTimer

Scope Operation

Control which Scope window signals are displayed, and how the sweep display operates.

Figure 4‑6. Step: Scope Operation

Default Setting: Scope, Wipe=false

Undo Removes any unsaved edits to this step.

Wipe Scope Clears the Scope window of sweeps

Persistence: No change

Off

Signal list: Enable to display a list of input signals

Add Signal Clear Clears the signal list

All Signals Selects all signals

[ List of available input signals ]

[ List of selected input signals ]

You can directly edit the list. User-defined signal labels can also be used.

Set Axis

Modify the Axis scaling of a signal.

Figure 4‑7. Step: Axis

Default Setting: Axis, Axis=Autoscale, Kind=Left, Target=Current1

Undo Removes any unsaved edits to this step.

[ Drop-down list ]

Autoscale Match the axis range to the data range

Autoscale from Zero Display from zero to the largest value

Full scale Display the full range of the axis

Use last Keep using the last-used settings

[ Drop-down list ]

Left Select Y-Axis Add a signal

Bottom Select X-Axis For all signals

Add Signal

Clear Clears the signal list

All Signals Selects all signals

[ List of all input signals ]

[ List of selected signals] Select an input signal to modify from a list and/or directly enter user-defined signal labels.

Set Checkbox

Set Checkbox uses simple “on/off” toggles. Checkbox status can be read by ‘If’’ and ‘ElseIf’ steps to make “yes/no” decisions and control the execution path of the Paradigm. If the equation evaluates to a non-zero value, the checkbox is enabled, i.e., ”on”.

Figure 4‑8. Step: Checkbox

Default Setting: Checkbox, Count=1, Equation=true

Undo Removes any unsaved edits to this step.

Checkbox Checkboxes [1 – 3] are local: they are cleared whenever a Paradigm is started.

Checkboxes [4 – 6] are global: they are never cleared, so their status persists across all Paradigms in the Experiment.

[Equation field] A free-form text field, evaluated to a value, and applied to the Checkbox.

[ Errors are reported under this field. ]

Check Equation Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports "Syntax is ok.”

· The constant “True” evaluates to ‘1.000’.

· The constant “False”evaluates to ‘0.0000’.

Insert special identifier Special functions for use in equations.

(See list below.)

Set Solution

A “solution” command is used to turn solution valves on or off in perfusion systems. A predefined digital pattern or analog level can be automatically output with this step. Solution settings are configured and numbered in the Solution Editor.

Figure 4‑9. Step: Set Solution

Default Setting: Solution, Target=Off

Undo Removes any unsaved edits to this step.

Set Solution Select a solution number to activate its valve. The number of available solutions depends on the Solution Editor configuration.

[ 1 – 16 ]

Set Tag

A comment tag can be automatically triggered with this step. Enter the optional tag text in its field. The last selection is used for all subsequent recordings within the same Paradigm.

Figure 4‑10. Step: Set Tag

Default Setting: SetTag, Text=

Undo Removes any unsaved edits to this step.

Tag text Enter the comment text.

During acquisition, a black vertical “tag” cursor is displayed in the Scope window at the tag time point, and time-stamped metadata entries are created.

If acquisition is not running when tagging occurs, metadata ‘Tag Comment’ entries are created during acquisition without time stamps.

Note: In emulation mode, tag timing is not accurate, and no tags are set in the first sweep.

Set Variable

Variables allow flexible control of any operation where equations are used, as well as Routine waveform amplitudes and durations.

Figure 4‑11. Step: Set Variable

Default Setting: SetVariable, Target=Paradigm, Count=1, Equation=p[1]

Undo Removes any unsaved edits to this step.

[ List of targets ]

· Paradigm Set the value of a Paradigm Variable_p[#]

[ 1 – 16 ]

· Paradigm_Input Set the value of the Paradigm Editor ‘Input’ control.

· <List of Routines> Select a Routine and modify its Routine Variable_r[#]

Select the Routine variable # (1 - 16).

Equation Evaluates to a value, used to set a variable or the Paradigm Editor ‘Input’ control.

To implement manual control of a variable, use the special identifier ‘Input’, which reads the Paradigm Editor ‘Input’ control. (See sample Paradigm ‘Tuning_with_Input’.)

Check Equation Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports "Syntax is ok.”

Insert special identifier Special functions for use in equations.

(See list below.)

Sound

Output a note from the computer speaker.

The frequency can be defined by a fixed value or an equation.

Figure 4‑12. Step: Sound

Default Setting: Sound, Equation=, Volume=1

Undo Removes any unsaved edits to this step.

Frequency (Hz) [ 250 – 8000 ]

Specify as an equation or fixed value.

The sound output has a linear frequency response range within its limits.

< 250 Hz: two clicks

> 8 kHz: 8 kHz tone

Check Equation Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports "Syntax is ok.”

Insert special identifier Special functions for use in equations (See list below)

Play Sound Test the sound output.

Volume [ 0.1 – 1.0 ]

Use the spinners for 10% increments, or directly edit the field.

Output is via the standard sound output that Igor uses:

· Windows: Built-in speakers, or a computer sound card with external speakers

Note: Lower frequency tones are attenuated in volume on lower-quality speakers

· macOS: Built-in speakers

This paradigm step can also be utilized as an Igor programming command. For instance, using an equation, one could listen to the membrane resistance of the cell under investigation

Example: Output a note.

Enter this equation in the Command window command line:

SutterPatch#Paradigm_PlaySound( 400, 1 )

View Last

Display the data from the last recording in a Scope (reanalysis) window.

Write Log

Enter text to be written to the Log window.

Figure 4‑13. Step: Write Log

Default Setting: WriteLog, Alert=true, Text=text_to_write, Equation=, DoBeep

Undo Removes any unsaved edits to this step.

Do Beep Generate a beep before writing.

Show Alert Display and/or edit the Alert text, then write it to the Log.

Run-time dialog

Elapsed time A time counter for the Alert

Text Edit the text message

Continue and Write Write to the Log

Continue, no Write Do not write to the Log

Stop Paradigm Halt Paradigm execution

Text to send to Log [ ]

Equation result to append [ ]

Check Equation Check the equation syntax for sweep #1. The equation is evaluated, and if valid, it reports "Syntax is ok.”

Insert special identifier Special functions for use in equations.

(See list below.)

Alert

Display an “Alert” dialog box that pauses Paradigm execution until manually dismissed.

Figure 4‑14. Step: Alert

Default Setting: Alert, Text=alert_text, DoBeep=true

Undo Removes any unsaved edits to this step.

Do Beep Sound a “beep” from the computer

Text to show in Alert Enter a message to the user.

Beep

Generate a “beep” sound from the computer speaker.

Default Setting: Beep

Comment

An in-line text message is created, and optionally displayed in a window.

Figure 4‑15. Step Comment

Default Setting: Comment, Text=

Undo Removes any unsaved edits to this step.

Comment Enter the comment text.

Show in window A floating ‘Paradigm Comment’ window is displayed with the comment text, and closes when the paradigm ends.

Wait

Temporarily pause execution of the Paradigm.

Figure 4‑16. Step: Wait

Default Setting: Wait, Time=1

Undo Removes any unsaved edits to this step.

Seconds to Wait The spinner increments in 0.1 s steps.

Break

Use a Break step to stop the execution of a Paradigm, or to interrupt For Loop and For Each Sweep loops.

Figure 4‑17. Step: Break

Default Setting: Break, Kind=Paradigm

Undo Removes any unsaved edits to this step.

Break Kind: Paradigm

ForLoop

Chain

Use to link step execution to another Paradigm.

Figure 4‑18. Step: Chain

Default Setting: Chain, Target=undefined_Paradigm, Return=true

Undo Removes any unsaved edits to this step.

Return to calling Paradigm: Once execution of the target Paradigm has completed, return execution to this Paradigm.

Paradigm to chain to: Paradigm execution will shift to the selected Paradigm.

Note: For multiple Chains (or recursive calls), you can call a maximum of eight Paradigms.

For Loop

Use a standard programming “For loop” to repeat a set of steps.

Figure 4‑19. Step: For Loop

Default Setting: ForLoop, Max=1

ForEnd

Undo Removes any unsaved edits to this step.

Max. ForLoop Count Number of loop cycles to run

Loop Indefinitely Sets ‘Max. ForLoop Count’ to ‘inf’

Note: A ‘For’ loop is processed as one step

Jump

Shift the Paradigm sequence to an arbitrary step. When executed, a jump occurs to the step after the target Label.

Figure 4‑20. Step: Jump

Default Setting: Jump, Target=jump_label

Undo Removes any unsaved edits to this step.

Jump Target Enter the Label of the step to jump to.

Label

Create a Label for a Jump step.

Figure 4‑21. Step: Label

Default Setting: Label, Target=jump_label

Undo Removes any unsaved edits to this step.

Label name Assign a name to the Label.

If

This step allows conditional Paradigm flow control between multiple choices.

Figure 4‑22. Step: If

Default Setting: If, Left=, Operation=’>’, Right=

EndIf

Undo Removes any unsaved edits to this step.

[ drop-down list ] Operation selection

· Compare 2 equations

Left Equation Evaluated to a value.

Check Equation Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports "Syntax is ok.”

Insert special identifier Special functions for use in equations.

(See list below.)

Operation: Comparison operators

> Greater than

>= Greater than or equal to

= Equal to

!= Not equal to

<= Less than or equal to

< Less than

Right Equation Evaluated to a value.

Check Equation Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports "Syntax is ok.”

Insert special identifier Special functions for use in equations.

(See list below.)

· Check for key pressed “Last key” typed on keyboard

Key to check for Enter a text key, or insert a “special” key

Insert special key Use a “non-text” key:

o Space

o Return

o Esc

· Check checkbox status

Checkbox Select a checkbox to monitor for “on/off” status.

Checkboxes are displayed at the bottom of the Paradigm Editor window.

[ 1 – 3 ] Paradigm-level “local” checkboxes

[ 4 – 6 ] Experiment-level “global” checkboxes

· Answer of yes-no-alert

Do Beep Your computer beeps once when the alert displays.

Alert Text [ ] Enter your alert question text.

Run-time dialog

Elapsed Time A time counter for the alert.

Yes ‘Yes’ button (value = 1)

No ‘No’ button (value = 0)

Stop Paradigm Manually abort the Paradigm

ElseIf

Allow conditional Paradigm flow control between multiple choices.

Figure 4‑23. Step: Else If

Default Setting: ElseIf, Left=, Operation=’>’, Right=

Undo Removes any unsaved edits to this step.

[ drop-down list ] Operation selection

· Compare 2 equations

Left Equation Evaluated to a value.

Check Equation Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports "Syntax is ok.”

Insert special identifier Special functions for use in equations.

(See list below.)

Operation Comparison operators

Greater than

>= Greater than or equal to

= Equal to

!= Not equal to

<= Less than or equal to

< Less than

Right Equation Evaluated to a value.

Check Equation Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports "Syntax is ok.”

Insert special identifier Special functions for use in equations.

(See list below.)

· Check for key pressed

Key to check for “Last key” typed on the keyboard

Insert special key Use a “non-text” key:

o Space

o Return

o Esc

· Check checkbox status Select a checkbox to monitor for “on/off” status .

Checkbox

[ 1 – 3 ] Paradigm-level “local” checkboxes

[ 4 – 6 ] Experiment-level “global” checkboxes

Checkboxes are displayed at the bottom of the Paradigm Editor window.

· Answer of “Yes/No” alert

Do Beep Your computer beeps once when the alert displays.

Alert Text [ ] Enter your alert question text.

Run-time dialog

Elapsed Time A timer of how long the Alert has been displayed.

Yes ‘Yes' button (value = 1)

No ‘No’ button (value = 0)

Stop Paradigm Manually abort the Paradigm

Else

This step allows Paradigm flow control to continue to the next step if the previous condition fails.

Default Setting: Else

Checkboxes Checkboxes are useful for quick conditional control of Paradigm steps. They are visible at the bottom of the Paradigm Editor window.

Figure 4‑24. Checkboxes

[ 1 – 3 ] These “local” checkboxes are cleared when a Paradigm starts. They provide Paradigm-specific controls that are only valid for the current Paradigm session.

[ 4 – 6 ] These “global” checkboxes are cleared when an Experiment starts. They can be used across all Paradigm Pools for the entire Experiment.

Input Routine and paradigm variables can be set to this value. ~~Enter~~ Manually enter a value, or set it via the paradigm step ‘Set Variable’.

[ -1.00 – 1.00 ] This value can be rescaled when used in an equation.

Paradigm Variables Pane You can Show/Hide a Paradigm Variables table at the bottom of the Paradigm Editor.

Figure 4‑25. Paradigm Variables

These variable can be used in any equation, or in the paradigm step Execute, and persist across experiments. The table can be directly edited during non-acquisition, or set via the paradigm step Set Variable.

‘Close’ button Closes the Variables table

Variable[1 – 16] 16 columns of Paradigm Variables

Name: Var_p[1 – 16] Paradigm Variable names can be edited to any text.

Note: These names are for display only, and cannot be used in equations.

Value: [ ] Numeric values can be manually entered, or programmatically set via the paradigm step ‘Set Variable’.

‘Insert Special Identifier’ List of special functions for use in equations.

loop (active paradigm ForLoop count)

sweep (active paradigm EachSweep count)

LastSweep (active paradigm sweep count of last sweep)

Processing can occur before or after the last sweep of a series.

Example: Use in a ‘ForEachSweep’ loop Routine, to compare an ‘If’ step equation to the sweep number.

ForEachSweep

EachSweep, Target=IV

If, Left=sweep, Operation= ‘=’, Right=LastSweep- 1

Alert, Text=LAST SWEEP, DoBeep=true

EndIf

ForEachEnd

AqStopped (last acquisition was stopped)

1= the last acquisition was stopped

0 = the last acquisition completed

ParadigmTime (time at start of paradigm, s)

RoutineTime (time at start of routine, s)

Stimulant (last applied stimulant concentration)

From the Solution Editor ‘Concentration’ setting, for a solution configured as a ‘Chemical Stimulant’.

time (present date-time, s)

timer (timer time, s)

---------

m[1..16] (n’th analysis measurement value)

gx[1..8] (n’th analysis graph x value)

The X-value of the last data point in the latest version of graph[#]

gy[1..8] (n’th analysis graph y value)

The Y-value of the last data point in the latest version of graph[#]

r[1..16] (n’th routine stimulus variable)

p[1..16] (n’th paradigm variable)

Hold[1..8] (holding of n’th output channel)

Input (Input variable on paradigm window)

--------

AuxIN[1..4] (reading of auxiliary input, V)

a single-point reading

Imon (amplifier current reading, A)

Vmon (amplifier voltage reading, V)

Mean[name or count,start,width] (mean of given input signal)

‘name’ = signal name

‘count’ = window-signal position

‘start’ = time of start, s (of measurement range)

‘width’ = duration, s (of measurement range)

--------

ActiveProbe (active probe)

The “active” probe is the IPA headstage [1 – 4] currently controlled by the Amplifier Control Panel. Most Paradigm Step commands apply to the active probe. For a single IPA amplifier, the headstage is always active probe ‘1’.

NumProbes (number of probes)

The total number of IPA headstages in the system.

--------

CCMode (amplifier current clamp)

VCMode (amplifier voltage clamp)

Hold (IHold in CC-mode, VHold in VC-mode) [in Routines]

[ ±0.000,020 A (±20,000 pA), or ±1.000 V (±1000 mV) ]

IHold (amplifer holding current, A)

[ ± 0.000,020 A (±20 nA) ]

VHold (amplifier holding voltage, V)

[ ±1.000 V (±1000 mV) ]

IHoldOn (amplifier holding current On)

VHoldOn (amplifier holding voltage On)

--------

IGain (amplifier current gain, V/A)

The gain of the active voltage-clamp ‘Current’ input channel is implemented in discrete levels:

0.5 mV/pA

1 mV/pA

2.5 mV/pA

5 mV/pA

10 mV/pA

25 mV/pA

Use a preset discrete level in an equation, or else a 90% threshold is used to promote values between the preset levels (to help avoid saturation of the input signal.)

VGain (amplifier voltage gain, V/V)

V/V evaluates to mV/mV.

The gain of the active current-clamp ‘Voltage’ input channel is implemented in discrete levels:

10 mV/mV

20 mV/mV

50 mV/mV

100 mV/mV

200 mV/mV

500 mV/mV

Use a preset level in an equation, or else a 90% threshold is used to promote values between the preset levels (to help avoid saturation of the input signal.)

Filter (amplifier input filter, Hz)

Apply a filter to the input channels.

Use a preset level in an equation, or a 10% threshold is used to promote values between the preset levels (to help avoid over-filtering).

500 (500 Hz)

1000 (1 kHz)

2000 (2 kHz)

5000 (5 kHz)

10000 (10 kHz)

20000 (20 kHz)

Offset (amplifier pipette offset, V)

OffsetLock (amplifier pipette offset lock On)

VTrack (amplifier tracking potential, V)

VTrackOn (amplifier tracking potential On)

--------

ECompMag (amplifier electrode compensation magnitude, F)

ECompPhase (amplifier electrode compensation phase, fraction)

ECompOn (amplifier electrode compensation On in CC-mode)

CmComp (amplifier cell compensation Cm, F)

RsComp (amplifier cell compensation Rs, Ohm)

RsCompOn (amplifier cell compensation Rs On)

RsLag (amplifier Rs correction lag, s)

RsPred (amplifier Rs prediction, fraction)

RsCorr (amplifier Rs correction, fraction)

RsCorrOn (amplifier Rs correction On)

Bridge (amplifier bridge balance, Ohm)

--------

Relectr (electrode/seal/access resistance, Ohm)

Value from last Membrane Test

Rmemb (membrane resistance (Cell mode), Ohm)

Value from last Membrane Test

Cmemb (membrane capacitance (Cell mode), F)

Value from last Membrane Test

4.1.5 (Scope) Real Time Measurements & Graphs

This special version of the ‘Real Time Measurements & Graphs’ dialog allows for quick interactive analysis changes during acquisition. Input channels, measurement regions, and graphing options can all be modified here. These changes instantly override the loaded Routine settings for quick interactive analysis, and can be saved back to the Routine.

Figure 4‑26. Acquisition Real Time Measurements & Graphs

To access, click on the Measurements button in the Scope (Acquisition) window.

Otherwise, this Scope version of the dialog is very similar to the Routine Editor version, with a few additions.

Extra buttons

Update Routine Pool The stored routine is updated with any changes

Revert to Original Any changes are reverted when the dialog is closed or the main routine is re-activated.

4.1.6 Routine Editor

Routines contain the settings that are in effect during data acquisition. The Routine Editor allows you to define acquisition parameters, set input and output channels, and to create stimulus waveforms and online analyses. The Routine Editor is the central place to create and manage saved Routine Pools and data acquisition settings.

Figure 4‑27. Routine Editor

The Routine Editor is structured to hold one or more Routines within its Routine Pool. The Routine Pool thus provides easy access to the set of Routines used in an experiment.

Tip: SutterPatch comes with a Sample Routine Pool that contains a collection of frequently used experimental scenarios. Rather than creating a new Routine, it might be easier to Duplicate a sample Routine and modify it until it meets your particular needs.

Status Field	Notifications on edits and Routine names are displayed here.
Files and Pools	[ drop-down list …]
	Most recently used list of the last 5 Routine Pool files
	New Routine Pool	Create a new Routine Pool either with a blank default Routine, or populated with Routines from the currently loaded Routine Pool.
	Load Routine Pool	Load the Routines of a previously saved Routine Pool file into the Routine Pool
	Revert to Last Saved	Undo any unsaved changes to the Routine Pool
	Save Routine Pool	Save the Routine Pool using its existing file name and path.
	Save Routine Pool as	Save the Routine Pool to a new file, and switch to the new file. The default file name has an increment number appended to the original file name.
	Save Routine Pool Copy	Save the Routine Pool to a new file, but do not switch to the new file. The default file name has ‘Copy of’ prepended to the original file name.
	Merge Routine Pools	Insert the Routines from a previously saved Routine Pool file into the loaded Routine Pool.
	Merge PatchMaster PGF File	Insert the “Routines” from a PatchMaster PGF file into the loaded Routine Pool.
	Convert Routine Pool	Convert the loaded Routines to be compatible with the attached amplifier or emulation mode. The original file is overwritten.

Table 4‑1: Files and Pools

New Routine Pool sample dialog

Figure 4‑28. New Routine Pool

[ File Path field ]

The file path of the current Routine Pool displays on the right of the ‘Files and Pools’ list. If the Routine Pool has not been previously saved, this field is blank.

Routine Description

A Routine Description text comment can be edited and saved with the Routine.

Routine Pool

The Routine Pool section lists the names of all currently loaded Routines. Selecting a Routine name loads it into the Routine Settings section for editing and activation. As the Routine Pool contents are held in memory, the switching times between Routines are very fast.

Double click a Routine name to edit it.

· Allowable characters are A-Z, a-z, 0-9, and “_”.

· Special characters and spaces are not allowed.

· The maximum length of a Routine name is 22 characters.

Note: When a Routine is selected, if it was created for a different system (i.e., IPA vs. Double IPA), and the Preferences / Hardware / Routine Conversion is set to ‘Prompt user’, then a Routine Conversion dialog displays.

Activate	Open or refresh the Scope window with the latest Routine settings, but do not start acquisition. This button displays as “In Progress” during a recording.
Execute	Open or refresh the Scope window and immediately start recording. The latest Routine settings are applied to the Scope window. This button displays as “Convert” if a routine designed for a different amplifier type is selected and the Hardware Preferences do not automatically convert it.
Save Pool	Save the Routine Pool using its existing file name.
New Routine	Add a blank Routine to the Routine Pool, and open it for editing. The default Routine name is “untitled” with an increment number appended.
Duplicate	Add a copy of the selected Routine to the Routine Pool. The Routine name number is appended or incremented.
Delete	Remove the selected Routine from the Routine Pool.
Revert	Discard any unsaved changes to the selected Routine.

Table 4‑2. Routine Editor Buttons

Waveform Preview

The stimulus waveform is graphically displayed at the bottom of the Routine Editor.

Figure 4‑29: Waveform Preview Pane

The waveform preview is updated live to reflect changes in the Waveform Editor and Amplifier Control Panel. It uses a “Cityscape” display mode, i.e. plotting with straight horizontal and vertical lines connecting the preview sample points (vs. the smooth interpolated transitions in recorded data).

The Preview Pane allows Measurement cursors to be manually dragged, and simultaneously updates their routine Start/End settings.

The preview for the digital output “DigOUTWord” sets its Y-axis to ‘ Digital State (Word)’, and displays the decimal value of the selected bits.

X- and Y-axis Control:

· Hover the mouse cursor over an axis line until the cursor turns into a double-headed arrow, then scroll up or down to contract/expand the axis.

· In the preview pane, click and drag the mouse cursor to surround a region of interest with a bounding box (the “marquee”). Right-click in the box and select one of the expand/shrink options.

Some key settings and display controls are listed on the right of the Preview pane:

Sweep Interval: The interval of time between the starts of consecutive triggered sweeps (Sweep Start-to-Start Time) in the active Routine.

When set to ‘Shortest’, this equals the longest Sweep Duration + 200 ms.

Sweep Duration: The amount of time in a sweep during which signal recording occurs with the active Routine.

Outputs enabled

The sweep duration is based upon the longest stimulus waveform duration set in Output Channels & Waveform / Waveform Editor.

Outputs disabled

The sweep duration is based upon the longest waveform duration set in Input Channels / Edit Signal / Waveform Editor.

Min. Stim. Duration: The minimum amount of time in which output stimulation occurs in a sweep.

Set in Output Channels & Waveform / Waveform Editor / Duration.

Max. Stim. Duration: The maximum amount of time in which output stimulation occurs in a sweep.

Set in Output Channels & Waveform / Waveform Editor / Duration.

Max. Stim. Points: The maximum number of stimulus sample points that are output in a sweep.

Show Sweep: Control the sweeps display in the preview pane (autoscaled).

· Sweep # a single sweep

· All Sweeps all sweeps overlaid from time zero

· Time Course: all sweeps in continuous linear time

Show Channel: A list of output channel to preview.

Show Measure Cursors: A list of measurement cursors to preview.

Routine Settings

The Routine Settings are split into 4 main sections. Click on a section header or its items to open its sub-window for editing.

Figure 4‑30. Routine Settings Sub-Window

Routine Editor: Acquisition & Routine Parameters

Acquisition timing parameters are controlled in this section, such as sweep duration and sampling rates.

The settings in this section are shared by all output channels.

Figure 4‑31. Acquisition & Routine Parameters

External Trigger Action

Control how and when recordings occur, using the external hardware digital input trigger (‘Trigger In’ BNC) on the back panel of the IPA amplifier.

· None: Hardware trigger inputs are disabled.

· Start Sweep: A trigger pulse starts recording of a Series that halts after one sweep, and then repeatedly waits for the next trigger pulse to record the next sweep, until the Routine is completed.

Acquisition Mode

· Triggered Sweeps: Standard sweep-oriented recording.

Each sweep is started either by an internal trigger from a Routine or Paradigm, or by an external trigger into the ‘TRIGGER IN’ connector on the back of the IPA amplifier. To account for triggering delays, there is a short gap (~200 ms) between sweeps.

· Continuous Sweeps: This option provides for continuous recording without any time gaps between sweeps, as all sweep outputs are concatenated together into a single output command wave.

Note: The data recording is displayed in a sweeps-oriented display (not a continuous or rolling display). Also, a command waveform is only generated for the first cycle

Enable outputs

Output channel waveforms are enabled by default, but can be optionally disabled.

When outputs are disabled, you can control the acquisition sweep duration via the Input Channels ‘Edit Signal’ button.

Number of Sweeps

The number of sweeps to record.

Sweep Cycles

The number of times to automatically repeat the entire set of sweeps to be recorded within a single Series.

Indefinite Record while continuously cycling through sweeps within the same Series. The recording continues until the Stop button is pressed.

Note: A command waveform is only generated for the first cycle in ‘Continuous Sweeps’ acquisition mode,

Sweep Start-to-Start Time (s)

The time from the start of recording a sweep to the start of the next sweep. This must be equal to or greater than the ‘Shortest Possible’ time.

Shortest Possible The longest sweep duration + 200 ms for overhead processing.

When disabled, the numeric value of ‘Shortest Possible’ is used as the default value.

Input Sampling Rate:

Frequency Sampling Interval

100 Hz (10 ms)

200 Hz (5 ms)

400 Hz (2.5 ms)

800 Hz (2 ms)

1 kHz (1 ms)

2 kHz (500 µs)

4 kHz (250 µs)

5 kHz (200 µs)

8 kHz (125 µs)

10 kHz (100 µs)

20 kHz (50 µs)

25 kHz (40 µs)

40 kHz (25 µs)

50 kHz (20 µs)

Tip: 10 kHz input channel sampling rates are standardly used, with 20 kHz occasionally used for faster events, and 5 kHz for slower events.

Note: Slow acquisition can take a long time to respond, due to 16k buffers to fill.

Output Sampling Rate:

Frequency Sampling Interval

100 Hz (10 ms)

200 Hz (5 ms)

400 Hz (2.5 ms)

800 Hz (2 ms)

1 kHz (1 ms)

2 kHz (500 µs)

4 kHz (250 µs)

5 kHz (200 µs)

8 kHz (125 µs)

10 kHz (100 µs)

Note: The sample Routines use a 1 kHz default output channel sampling rate, as command waveforms usually do not require high resolution time changes - increase this setting as needed.

Persistence Display

For a recording, display each new sweep without erasing any previous sweeps.

· Off Set the Scope window into non-persistence display mode.

· On Set the Scope window into persistence display mode.

· Keep current setting Do not change the Scope window’s prior setting.

Routine Editor: Input Channels

Configure the input channels.

Figure 4‑32. Input Channels

[ Status field ] Notifications on edits are displayed.

[ HW Status field ] Hardware information on the selected channel is displayed.

Channel

Enable input channels to be digitized and recorded from:

Current# Analog input current channels hardwired from the IPA headstage

Voltage# Analog input voltage channels hardwired from the IPA headstage.

AuxIN[1 – 4] Four auxiliary analog input channels allow you to directly digitize and record input signals from connected non-Sutter external equipment.

Note: In Emulation mode, the AuxIN channels display a ±20 mV sine wave.

Virtual[ 1 – 10 ] Ten Virtual channels are available.

Virtual channel data are mathematically transformed data from another input channel, or are entirely computed from an equation.

Label

A user-defined signal name for a channel.

These labels are used in:

· ‘Parent Out Chan’

· Virtual channel Math Equations and Source Channels

· Scope window signal panes

· Data Navigator Preview pane

· Metadata: Input Signal Name

To rename an Input Channel, first enable it, then double-click it and enter the new name. If the label is used by another channel, an underscore and increment number are appended to the new label.

Note: If a Virtual input channel is set to ‘Math Type: Leak’, a default ‘LeakSubtracted’ label is automatically created.

Input Unit The base unit of measurement.

· ‘Current’ Channels Fixed at ‘A’ for current.

· ‘Voltage’ Channels Fixed at ‘V’ for voltage.

· ‘AuxIN’ Channels Default is ‘V’, but is editable.

· ‘Virtual’ Channels Default set from its Source channel, but is editable.

Note: The resolution of the signal units are automatically set.

Scaling

Offset Apply an amplitude offset to an Auxiliary input signal

(after any scaling).

Tip: To use ‘mV’ units, enter: ‘#m’ or ‘#e-3’

To use ‘pA’ units, enter: ‘#p’ or ‘#e-12’

Factor

Apply a scaling factor as a fixed value or as an equation.

Note: The IPA digitizer uses a high-resolution 16-bit ADC with 64-bit data, so data resolution is not an issue when scaling input signals.

Parent Out Chan

The “Parent Output Channel” is an advanced feature to support Segment-based measurement timing patterns.

When measurements are configured with ‘Cursors relative to: Segment’, you can select which ‘Parent Out Chan’ (Output Channel) segment timing information is to be used by the Input Channel measurement cursors.

Virtual channels cannot directly select Parent Output channels. Instead, they utilize their Source channel Parent channels.

Scope Position The Input Channel signal panes can be repositioned in the Scope window.

Edit Signal This button opens a simplified Waveform Editor to allow modifying segment timing for measurements. You can also open the simplified Waveform Editor by double-clicking on a highlighted Input Channel name.

When outputs are disabled in the Acquisition & Routine Parameters section, the main Waveform Editor is also disabled in the Output Channels & Waveform section, and the Edit Signal button is enabled.

Virtual Input Channels

If a Virtual input channel is enabled, its configuration fields are ungrayed:

Math Type Apply a data transformation to a virtual input channel.

Leak Remove leakage current from the data.

Source channel Select an input channel to transform.

Show leak Displays the accumulated leak currents after the subtracted data in a sweep.

Leak zero segment Identify a segment with no active cellular response to the command signal.

Note: The mean of the second half of the specified segment is used to compute an averaged leak current, which is then used to correct the P/N leak average. This option reduces the influence of a constant leak-current, which is otherwise added to the leak current of the main pulse.

Smooth Smooth the data with a “moving average” noise-reduction filter

Source channel Select an input channel to smooth

Smoothing type:

· Gausian A standard filter with exellent 10 – 90% rise-time response.

Smooth operations [ 1 – 32767 ]

# of smoothing operations to perform

· Boxcar A fast time-domain filter with excellent 0 – 100% rise-time response

Smooth repetitions [ 1 – 32767 ]

# of smoothing repetitions to perform

Boxcar window points [ 1 – 99 ] (odd values only)

# of points in boxcar window

LineFreq Remove AC line frequency noise (hum) from the data.

Source channel Select an input channel to apply noise reduction to.

Line Frequency 50 Hz

60 Hz

Alternating current (AC) power contains 50 or 60 Hz oscillations that can cause sinusoidal line-frequency noise in recorded signals. This ~~feature~~ FFT-based filter reduces such noise by ~> 90% over 6 harmonics. The adjusted signal is displayed in real time.

Equation Specify a mathematical equation to create virtual data.

(See the Equation Editor for more details.)

[ Equation field ] A free-form text field

[ Errors are reported under this field. ]

Note: The full equation is always visible as a tool tip - hover the mouse cursor over the Equation field.

Check Equation Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports that the syntax is “ok”.

Insert special identifier

· t[#] n’th input trace

The trace wave of the channel in Scope Position "n" is accessed using this substitute for the data folder path.

You can duplicate a trace by using a virtual signal of Math type "equation", using the equation "t[1]" for the first Scope signal.

· p[#] n’th paradigm variable

Undo All changes in the equation editing session are discarded.

Stimulus Replicate the Command Waveform

Source channel Select an input channel – the waveform from its ‘Parent Out Chan’ is used.

Differentiate Data transformation using differentiation

Source channel Select an input channel to differentiate.

Integrate Data transformation using integration

Source channel Select an input channel to integrate.

~~BesselFilter A frequency-domain filter~~

~~Source channel Select an input channel to filter.~~

~~Filter Coefficients Select a frequency range~~

· ~~LowPass Allow signal frequencies < the cutoff frequency~~

· ~~HighPass Allow signal frequencies > the cutoff frequency~~

~~Filter Order Number of “poles” in the filter~~

~~[ 1, 2, 4 ]~~

~~Cutoff Frequency (Hz) Restrict frequencies from this boundary point onwards~~

~~[ 100 to < ½ the sampling rate ]~~

~~LockIn~~

~~Source channel Select an input channel~~

~~Trace kind~~

~~RealY~~

~~ImagY~~

~~AdmitY~~

~~Phase~~

~~RealZ~~

~~ImagZ~~

~~ImpZ~~

~~Points to average~~

~~[ 1 – 10 ]~~

Routine Editor: Output Channels & Waveform

Configure the output channels and command waveform behavior.

Figure 4‑33. Output Channels & Waveform

[ HW Status field ] Hardware information on the selected channel is displayed.

Digital Outputs set as

Eight digital output channels (bits) can be set individually or as a group.

· Individual bits Each DigOUT(1 – 8) bit is individually set in its own Waveform Editor table.

The waveform preview uses the bit’s binary word value for its Y-axis value, i.e. if bit 3 is ‘HIGH’, it has a ”word” value of 4.

· 8-bit Word The 8-bit digital output pattern is controlled by a single decimal number (0 - 250), which is also the waveform preview amplitude value.

The waveform preview uses the binary bit pattern word value for its Y-axis value, i.e., if bits ‘1’ and ‘3’ are ‘HIGH’, the 8-bit word has a value of 5.

Channel

Click on the Output Channel checkboxes to enable analog and digital output channels in the Routine. Click on a channel name to highlight and select it – the channel output parameters are displayed for configuration.

The default StimOUT channnels are hardwired to the IPA headstage.

The two rear panel auxiliary analog output channels (AuxOUT1 & 2) can be used to send stimulus waveforms to external instruments.

The digital outputs are available as either a single 8-bit “word”, or as 8 individual 1-bit channels, as set in the Acquisition & Parameters section.

To rename any of the Output Channels, double-click on the respective name and enter a new one.

Tip: If a signal is connected to the front panel ‘COMMAND IN’ BNC, that signal is summed with the StimOUT waveform that is sent to the headstage.

Note: For StimOUT channels, the actual DAC output signal is passed through a 20 kHz low-pass filter before entering the headstage.

Label

A user-defined signal name for the channel.

These are used in:

· ‘Copy Channel’

· Waveform Preview pane ‘Show Channel’

· Metadata: Output Signal Name

To rename an Output Channel, first enable it, then double-click it and enter the new name. If the same label is reused for another channel, an underscore and increment number will be appended to the new label.

Copy channel

Copies one channel waveform to another channel, but only works between output channels of the same type (i.e., “Stim”, “Aux”, or Digital). If a channel is enabled, then highlighting another or blank channel of the same type ungrays its ‘Copy channel’ field, and changes it from ‘OFF’ to ‘None’, with a drop-down list of available channels to copy from.

Edit Waveform

Click the Edit Waveform button (or double-click on a highlighted Output Channel name) to access the Waveform Editor table and create a stimulus waveform.

(See the Waveform Editor section below.)

Restrict to

The routine’s IPA headstage StimOUT channels have to match the VC/CC mode of the IPA amplifier, or else the Routine cannot be activated or executed.

The default setting for new routines is ‘VC Mode’. This prevents CC mode pA (10^-12A) current outputs from being accidentally overscaled by VC mode routines using mV (10^-3V) voltage outputs.

· VC Mode The Amplifier Control Panel must be in VC mode to run this routine

· CC Mode The Amplfier Control Panel must be in CC mode to run this routine

Note: The IPA amplifier can be switched into any mode (VC or CC) while a recording is in progress, however it is your own responsibility to interpret data from mixed recording modes.

Output Unit

Enter the base unit of measurement. The signal unit resolution is automatically adjusted.

· StimOUT Channels

Fixed at ‘V’ for voltage clamp and “other” experiment types; fixed at ‘A’ for current clamp experiments.

· AuxOUT Channels

Set at ‘V’, but can be edited to any setting.

Scaling

Offset Apply an offset to the Auxiliary output channel (after any scaling.)

Factor Apply a scaling factor as a fixed value or an equation.

(See the Equation Editor for more details.)

Relative to Holding

If ‘Relative to Holding’ is enabled, the output signal is the command waveform summed with the ‘Holding’ level in the Amplifier Control Panel. If the Holding level is set to ‘0’, this setting has no effect.

Note: This setting is updated live by holding level changes in the Amplifier Control Panel.

Return to Holding at Sweep End

During a Series, set the output signal to the holding level while the system is not acquiring data, i.e., the time between sweeps after a sweep acquisition ends, but before the the next sweep starts.

This ensures that your cells are kept in a resting state as much as possible, and that each output sweep starts from the same holding level.

When ‘Return to Holding at Sweep End’ is not enabled, the output signal uses the last value of the command waveform during the time between sweeps, and returns to the original holding potential (as shown on the Amplifier Control Panel) when the routine stops.

Note: You might want to avoid returning to an unwanted holding level at the end of a series, as such a situation could send a short but disruptive glitch to your preparation. To avoid this, create a Paradigm and set a new target holding level in an "Each Sweep" loop just before the last sweep is started.

This setting is updated live by holding level changes in the Amplifier Control Panel if ‘Relative to Holding’ is enabled, otherwise it is only updated at the time of routine activation.

Enable P/N Leak Pulses

Endogenous leak currents can flow, even while a cell is in its resting state, from conditions such as an imperfect or leaky seal, or via existing ion channels, and affect response amplitudes. Online P/N leak subtraction automates the removal of such currents from the data.

If endogenous leak conductance is an issue with your cell type, and/or high temporal resolution is required along with a need to reduce capacitive transients (e.g., with voltage-gated sodium currents), click ‘Enable P/N Leak Pulses’ and configure its settings below.

A “leak pulse” is a replica of the stimulus waveform, and is used to record a fraction of the leakage current. In this technique, leak pulses are generated, and the responses are averaged, scaled, and subtracted from the main response to remove the effects of leakage.

Note: The sub-pulses are stored as part of the sweep. This ensures that if any events occur during the sub-pulses or between the sub- and main pulses, which leads to unexpected or hard-to-interpret effects, the full original recording condition can be examined.

Preview Leak Pulses

Display the leak subtraction pulses in the Routine Editor Waveform Preview panel. A leak subtraction pulse is a scaled copy of the main stimulus waveform.

Alternating Leak Polarity

You can reduce directional bias in the leak conductance by alternating the polarity of the leak subtraction pulses on a sweep-by-sweep basis, as long as no ion channels are activated.

Num Leak Pulses

Set the number of leak pulses used to average out noise and leak conductance. Adjust this number in accordance with the amount of noise in the signal. With the high precision of modern 16-bit digitizers, this number can sometimes be reduced to less than 4 leak sub-pulses.

Note: As each leak pulse replicates the stimulus waveform, larger numbers of leak pulses is not recommended, as this can greatly increase the total duration of a sweep during acquisition, and the noise in the sub- and main pulses can add up and actually increase.

The default setting of ‘4’ Leak Pulses, when used with the default Leak Ratio (-0.250) operates equivalently to pCLAMP’s default P/N setting (4 subsweeps for P/4).

Leak Ratio

Set the leak subtraction pulse size relative to the main waveform pulse, using a ratio between +1 and -1. The setting should be low enough that no electrically-gated ion channels are activated. For instance, a Leak Ratio setting of 0.25 will generate leak pulses at ¼ the amplitude of the main stimulus waveform, while a Leak Ratio of 0.2 will generate leak pulses at 1/5 the main pulse amplitude.

Note: The program scales the leak subtraction pulses based upon the Leak Ratio setting, not the number of Leak Pulses. This means that the Leak Ratio can be set independently from Num Leak Pulses, instead of those settings being interdependent.

Tip: As an alternate way to avoid electrical activation of ion channels, use a negative ratio to reverse the polarity of the leak pulses relative to the main pulse.

Leak Hold

The leak pulses holding level can be set differently from the Routine main holding level, for flexibility in finding a suitable leak pulse voltage range. The scaled waveform amplitudes are relative to the Leak Hold level instead of to the IPA holding level.

Set to a fixed value, or enter as an equation.

(See the Equation Editor section for more details.)

Check Equations Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports that the syntax is “ok”.

Insert special identifier Special functions for use in equations.

Undo All changes in the equation editing session are discarded.

Leak Pulse parameters common to all D/A channels

Leak Delay (ms)

If a settling time is needed between the leak pulses and the main waveform pulse, Leak Delay will insert a time delay between the execution of the leak pulses and the main pulse. Provide enough time to avoid interference of the leak pulses with any active currents or inactivation of ion channels.

When leak pulses occur before the main pulse, Leak Delay uses the IPA Holding level; when leak pulses occur after the main pulse, Leak Delay uses the Leak Pulses ‘Leak Hold’ level.

Enable A/C Line Frequency adjustment

The effect of AC line-frequency noise (hum) can be automatically reduced during P/N leak subtraction recordings.

50 Hz Enable the reduction of 50 cycle AC line noise.

60 Hz Enable the reduction of 60 cycle AC line noise.

This Line Frequency adjustment automatically calculates the proper interpulse interval for the P/N pulses, so that they are counter-phased to the line frequency of the output signal, which reduces hum without filtering the signal.

Execute Leak Pulses

The leak pulses can be set to run before or after the main waveform pulse.

· Before Main Pulse Sub-pulses are output relative to the Leak Hold level. After the sub-pulses complete, the signal goes to the IPA Holding level for the duration of the Leak Delay before the main pulse.

· After Main Pulse After the main pulse completes, the signal goes to the Leak Hold level for the duration of the Leak Delay setting, and then outputs sub-pulses relative to the Leak Hold level.

Routine Editor: Waveform Editor

Click the ‘Edit Waveform’ button to open the Waveform Editor and design a command waveform for the selected output channel.

Figure 4‑34. Waveform Editor

Close button Use this button to close the Waveform Editor window.

Used segments: # out of 50 Up to 50 contiguous segments can be configured in a waveform.

Actions

OFF Unused segments are labeled as ‘OFF’.

Tip: A segment with a Duration of ‘0’ ms is equivalent to ‘OFF’. This is a convenient way to skip a segment instead of deleting it.

Stored Enable a segment for stimulation and recording.

Not In Leak If P/N LeakPulses are enabled, this will optionally exclude the segment from being generated in the P/N Leak Subtraction output wave.

This is useful for inactivation or recovery studies, when commands do not change for long periods of time.

Insert Insert a default Segment into the current position, and increment the position of the following Segments, i.e. move them to the right.

Copy To copy a segment, click the segment’s Actions list and select ‘Copy’. A copy is inserted as the next segment.

To copy multiple segments, select the segments to be copied. Then, for the segment to be inserted before, click its Actions list, select ‘Copy’, and enter the number of times to copy the segments - the selected segments are inserted before the “Copy” segment.

Delete To remove a segment, select its ‘Delete’ Action.

Note: If there is only one segment, it cannot be deleted - there is always at least one segment enabled.

To remove multiple segments, select the desired segments. Then, click any segment’s Actions list and select ‘Delete’. All selected segments are deleted.

Tip: To select multiple segments, use the Windows CTRL-click / macOS ⌘-click to highlight segments, or SHIFT-click to highlight a range of segments.

Any following segments shift their Segment #’s down by the number of deleted segments.

Waveform Select the waveform shape.

Step The waveform amplitude rapidly jumps from a pre-existing level to the new level within one sample point, and stays at the new level for the duration of the segment. The resulting waveform shape looks like a step.

Ramp The waveform amplitude goes from the previous amplitude to the new amplitude as a smooth straight line - the sloping line looks like a ramp.

Tip: To create a sawtooth pattern, follow a ramp segment with a zero duration Step segment. This sharply resets the ramp amplitude to the baseline level – the next ramp will start from the baseline level again.

Sine The waveform is a sinusoidal wave.

Sine Wave Cycles Multiple One or more cycles

Single One cycle

Note: For a single cycle,the Cycle Duration is equal to the Segment Duration.

Cycle Amplitude First peak amplitude from baseline

Tip: To offset a sine wave from the default baseline (0 units), set the segment Amplitude, or enable Routine Editor / Output Channel ‘Relative to Holding’.

Cycle Duration One cycle length (ms)

Segment Duration Sine wave duration (ms)

Live Preview The Waveform Preview is updated in real time. If computer resources are low, this feature can be disabled.

Squarewave The waveform generates a train of rectangular pulses

Base Amplitude Increment Increment the baseline amplitude for each succesive pulse

Step1 Amplitude Amplitude of first pulse

Step1 Width Duration of first pulse (ms)

Step2 Amplitude Amplitude of second pulse

Step2 Width Duration of second pulse (ms)

Segment Duration Square wave duration (ms)

Live Preview The Waveform Preview is updated in real time. If computer resources are low, this feature can be disabled.

Chirp This waveform generates a sinusoidal wave that changes its frequency over time.

Chirp Type Linear A linear change in frequency

Geometric A geometric change in frequency

Note: For a geometric chirp, a minimal frequency range is enforced: the End Frequency has to be at least double the Start Frequency, or half or less than the Start Frequency.

Amplitude [ ±1000.00 mV, ±20000.00 pA ]

Start Frequency [ 1 – 50000 Hz]

End Frequency [ 1 – 50000 Hz ]

Segment Duration Chirp wave duration (ms)

Live Preview The Waveform Preview is updated in real time. If computer resources are low, this feature can be disabled.

Template Assign arbitrary waveforms to a segment.

Figure 4‑35. Template Waves

[ Status field ] The Routine segment # and output sampling rate are displayed

If the template sampling rate does not match a Routine sampling rate, the template is interpolated to match the Routine sampling rate.

Template Pool Templates loaded in the Template Editor Pool plus extracted templates.

Copy the selected template wave from the Template Pool into a Routine and Segment.

Copy the selected template wave from the Template Pool into a Routine.

In Routine Templates stored in the Routine.

Each output channel can have a maximum of 16 template waves stored in its routine. The same template can be used in multiple segments. Each segment can have one or multiple templates assigned to it.

Probably the most used case will simply be a single template paired with a single segment, but the possibilities are endless.

Note: To avoid unnecessary increase in the sizes of routines and Routine Pool files, only include templates that are actually used by segments.

Duration (ms) The duration of the template trace.

Copy the selected template from a Routine to a Segment.

Delete the template wave from a Segment or an unused Routine

In Segment Each segment can have one or multiple templates assigned to it.

If only one template is listed, then for any number of sweeps, the segment output wave will be the same for all sweeps.

If more than one template is assigned to a segment, and the number of sweeps is greater than 1, then an output wave is created as:

1st sweep with the 1st template on the list,

2nd sweep with the 2nd template,

etc.

If there are more sweeps than templates, then the templates are reused (round robin) for each subsequent sweep.

Set segment duration Set the segment duration to match the selected Segment template duration (also shown in the Routine Duration.)

Segment Duration (ms) The Segment Duration can be manually adjusted here.

Live Preview The Waveform Preview is updated in real time. If computer resources are low, this feature can be disabled.

Amplitude (analog) Select the waveform amplitude for a Segment. For the Sine, Squarewave and Chirp waveforms, this is used as an initial offset.

Set to Hold Use the Amplifier Control Panel holding level for the Segment amplitude.

Tip: When used in Segment 1 for voltage-clamp experiments, this allows you to record the leak current along with the actual holding voltage. For current-clamp experiments, this allows you to record the actual cell potential along with the actual holding current.

Avoid using the last Segment for this, as post-stimulation data can be recorded, such as from tail currents.

Value Use a single number for the segment amplitude.

Value List Use an arbitrary segment amplitude for each sweep.

For each sweep, enter a number or equation. Use the keyboard “up/down arrows” to edit another row.

Value+Increment Increment the segment amplitude for each sweep.

Start value [ ±1000.00 mV, ± 20000 pA]

Operator [ +, - ]

Increment value [ ±1000.00 mV, ± 20000 pA]

Segment Duration Same as the main Segment Duration

Live Preview The Waveform Preview is updated in real time. If computer resources are low, this feature can be disabled.

Equation Specify the segment amplitude as an equation.

[ Equation field ] A free-form text field.

Errors are reported under this field.

Check Equations Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports that the syntax is “ok”.

Insert special identifier Special functions for use in equations.

Undo All changes in the equation editing session are discarded.

(See the Equation Editor for more details.)

Var_r[ 1 – 16 ] Variable labels are displayed if the Routine Variables table is enabled.

Amplitude (digital) Digital settings are displayed if digital outputs are enabled.

Bit Set a steady-state digital level for an individual bit.

§ LOW = O 0

§ HIGH = 1

Bit Word Values are the decimal number of an 8-bit word (0 – 255), also displayed as a bit pattern.

Duration Set the segment duration.

Set to Hold Use the Amplifier Control Panel to set the I/O Digital Output holding levels.

Value Use a single number for the segment duration.

[ 0 – 2e+09 ms ]

Value List Use an arbitrary segment duration for each sweep, from a list of arbitrary numbers.

For each sweep, enter a row number or equation. Click the keyboard “up/down arrows” to edit another row.

(Blank lines are removed.)

Value+Increment Increment the segment duration for each sweep.

Start value [ 0 – 2e+09 ms ]

Operator [ +, - ]

Increment value [ 0 – 2e+09 ms ]

Live Preview The Waveform Preview is updated in real time. If computer resources are low, this feature can be disabled.

Equation

Specify segment duration as an equation

[ Equation field ] A free-form text field

[ Errors are reported under this field. ]

Check Equation Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports that the syntax is “ok”.

Insert special identifier Special functions for use in equations.

Undo All changes in the equation editing session are discarded

(See the Equation Editor for more details.)

Var_r[ 1 – 16 ] Variable labels are displayed if the Routine Variables table is enabled.

Segment Selection

Left-click Selecting an active segment turns its background gold, and enables the Actions / Copy and Delete functions. This allows multiple segments to be copied or deleted at the same time.

Tip: Standard mouse behaviour is used to select segments, i.e., Windows CTRL-click / macOS ⌘-click selects individual segments, and SHIFT-click selects a range of segments.

Routine Editor: Real Time Measurements & Graphs

Online analyses are configured in the Real Time Measurements & Graphs dialog. Measurement regions display in the Scope window, and their associated analyses are plotted in an Analysis sub-window during acquisition.

Figure 4‑36. Real Time Measurements & Graphs

For instant interactive analysis changes during acquisition, click the Scope window Measurements button, which opens a special version of the Real Time Measurements & Graphs dialog.

Disable Execution of Measurements and Graphs Block all measurements and analyses with one click.

[ Status field ] A short description of the selected Analysis.

On Enable an analysis to run.

ID Measurement regions are identified with an ID number: m[#]

[ 1 – 16 ]

Label Replace the default measurement ID with your own measurement name, to display in the Waveform Preview and Scope windows, and for use in equations.

Analysis Choose from 16 predefined Analysis statistics for each measurement:

Mean Arithmetic mean of the samples

Max value Value of largest sample

Time of max Time from sweep start to largest sample

Min value Value of smallest sample

Time of min Time from sweep start to minimum sample

Absolute peak Largest absolute value

Time of absolute peak Time from sweep start to largest absolute value

Rise time 10 - 90% rise time

Decay time 10 - 90% decay (fall) time

Slope Slope of linear fit

Std deviation Standard deviation of the samples: √(variance)

Variance Variance of the samples

Area Signed area (negative values negate positive values)

Absolute area Absolute area (negative values are converted to positive)

Frequency Number of threshold crossings per second (Hz)

Time to threshold Time from sweep start to first threshold crossing

Rise Tau Time constant of rise time

Decay Tau Time constant of decay time

Weighted tau Weighted time constant

RMS noise Root-Mean-Square noise

Segment duration Duration of the selected segment

Segment amplitude Maximum amplitude of the selected segment

These analyses can be directly plotted, or used in more complex equations.

(See the Equation Editor section for more details.)

Signals For each enabled Analysis measurement, select the signal to be measured from the list of Input Channels. A measurement is only made on one input channel, but it can be used in multiple graphs.

Cursors Relative to Set the measurement boundaries with left/right cursors.

· Trace Encompasses the entire trace.

Width: # s The width of the cursors in seconds.

· Sweep Time Set relative to the start time of a sweep (time zero).

Start time Set the left cursor relative to the Sweep start time (s).

End time Set the right cursor relative to the Sweep start time (s)

Note: Cursor Start times are always before their End times.

Step Cursors by: [ # Sample (# unit)]

Set the step size of the ‘Start time’ and ‘End time’ spinners (up to 50 samples.)

Lock step width Fix the width of the measurement region.

The measurement width is maintained at a constant value when the ‘Start time’ is updated.

Width: # s The width of the cursors in seconds. The minimum width size is 2 sample points.

· Segment #

Set relative to the start time of a Segment #.

Uses the segment timing from the input signal’s “Parent Output Channel”.

Start ratio [ 0 = beginning of Segment ]

Set the left cursor as a ratio of the Segment duration.

End ratio [ 1 = end of Segment ]

Set the right cursor as a ratio of the Segment duration.

Note: If the Start/End ratios extend past the boundary of a Segment, and the measurement is switched to a beginning or ending Segment, the Start/End ratios are reset to ‘0’ and ‘1’ respectively.

Lock step width Fix the relative width of the measurement region.

The measurement width is maintained at a constant ratio value when ‘Start ratio’ is updated.

Width: # s The width of the cursors in seconds. The minimum width size is 2 sample points.

Threshold This amplitude level needs to be crossed by the data to trigger measurements for:

· Rise/Decay time

· Rise/Decay Tau

· Frequency

· Time to threshold

Polarity The direction of a Threshold crossing.

· Positive Positive direction threshold crossing.

· Negative Negative direction threshold crossing.

· Largest Change Use the polarity direction of the largest change. (For Rise / Decay analyses only.)

Smoothing Factor Smoothen noisy data to reduce the effects of high-frequency noise on measurements by averaging the data sample points with an unweighted sliding average.

[ 2 – 200 ]

Note: Smoothing is not applied to the analyses ‘Segment duration’ and ‘Segment amplitude’. These are fixed values not subject to modification.

Enable When disabled, this field is set to ‘OFF’ (i.e. zero); when re-enabled, this field is reset to a value of ‘2’.

Graphs

On Enable a graph to configure its settings.

ID Graphs have a default ID (identification): g[1] - g[8]

Show Display this graph in an Analysis window during acquisition and analysis.

Note: If the Y-Axis ‘Equation’ field is blank, the graph will also be blank.

Label Double-click and enter a graph label to display in an Analysis graph.

Copy Use to transfer graph settings to a new graph. Highlight a disabled Graph ID, (but do not enable it), then select from the drop-down list of enabled graphs.

Y-Axis Select a measurement ID to plot against the graph Y-Axis, or select a Y-Equation for a customized Y-axis plot.

Label Enter a customized name for the graph Y-Axis.

Equation Click to edit with the Equation Editor.

For example, to monitor cell compensation, use the Equation Editor Special ID “RsComp”.

(See the Equation Editor for details.)

If running from a planned Paradigm, see the “For Each” step example to track cell changes sweep-by-sweep.

Unit Select a standard unit from the drop-down list, or enter a custom unit type.

Note: Standard unit resolutions, such as ‘pA’ or ‘mV’, are automatically calculated and displayed in the graph.

X-Axis Display the graph X-axis with a standard Time base, or select a measurement ID to plot against the X-axis, or use an X-Equation for a customized X-axis plot.

Label Enter a customized name for the X-Axis

Equation Click to edit with the Equation Editor

(See the Equation Editor for details.)

Unit Select a standard unit from the drop-down list, or enter a custom unit type.

Note: Standard unit resolutions, such as ‘pA’ or ‘mV’, are automatically calculated and displayed in the graph.

Routine Editor: Routine Variables

Up to 16 Routine Variables can be configured for stimulus control. These variables allow interactive manual control of the waveform amplitude and duration, or automatically via Paradigms.

Figure 4‑37. Routine Variables

Routine Variables

· Disabled

· Enabled Once Routine Variables are enabled, they become visible in the Waveform Editor Amplitude and Duration lists.

In Use A checkmark means this Routine Variable is in use by the Waveform Editor.

Label Var_r[ 1 – 16 ] Edit the default variable name if desired.

Value Numeric values can be manually entered or set by a Paradigm.

Note: If Routine Variables are enabled and then disabled, the Waveform Editor converts its ‘Var_r’ settings to ‘Value’ settings using the last enabled values.

4.1.7 Scope (Acquisition)

Scope windows provide the central view of your data, whether for data acquisition or data analysis. The data is displayed as a smooth interpolated line. While many of the window controls are common to all Scope windows, each type of Scope window has its own set of buttons.

Figure 4‑38 Scope Acquisition Window

Note: Only one Scope window can be open at a time. For example, if a Scope window is open for data acquisition, then opening it for the Membrane Test, Free-Run, or data analysis, will close the acquisition Scope window and re-open it as the new type of Scope window.

The Scope window is titled with the active Routine name.

Controls

Signals The central area of the Scope window graphically displays data in up to 16 signal panes. There is a separate pane for each enabled input signal. The “active” pane receives the focus of the Y-axis controls (magnify, scroll), and its border area is displayed in a lighter color. When multiple panes are displayed, the inactive panes are displayed with darker axis border areas. Click on a signal pane to make it active.

If multiple signals are displayed stacked on top of each other, their panes can be vertically resized by clicking and dragging the horizontal area between panes, using the resizing mouse cursor (a horizontal line with a vertical double-headed arrow.) The standard mouse cursor will change to the resizing cursor when positioned over a pane separator.

Note: Two additional data points are appended to the sweep data to support post-sweep holding levels and segment boundary rounding issues.

Signal Display Mode

Figure 4‑39. Signal Display Mode

Signal pane arrangements are set by the ‘Select signal display mode’ button in the lower left corner of the Scope window. Select how the input signals are to be graphically arranged:

· Stack Vertical column of signals

· Single Only the active signal

· [ 1 x 2 ] Tiled configuration of signals in rows and columns

Cursors Vertical measurement regions can be set up for input signals. Measurement regions display as light gray vertical bars in the signal panes. Each measurement region has a measurement width, bounded by a start-time cursor and an end-time cursor - cursors are the left and right edges of these regions.

To graphically move a measurement region, click and drag them with the mouse (when selected, the region turns dark.)

To resize a measurement region, click and drag a start- or end-time cursor (the left or right edge of a region.)

Magnification and Scrolling

Signals can be magnified or unmagnified using several X- and Y-axis display controls in the Scope window. Any magnification applied to the signals persists during acquisition.

§ Magnification Combo

Click on the “+” and “-“ buttons to magnify/unmagnify by steps, or click and drag the slider to smoothly zoom/unzoom the active signal. The X- and Y-axes have their own combo display controls. However, the Y-axis magnification only controls the active pane.

§ Axis Zoom

Figure 4‑40. Axis magnification

§ Area Zoom:

An area in a signal pane, such as an interesting part of the data, can be arbitrarily selected and expanded.

1. Move the mouse cursor into a signal pane, and it changes into a large “+”.

2. Click and drag a bounding box around a region of interest. The box is also referred to as a “marquee”.

3. Right-click in the marquee and select ‘Expand’ or ‘Shrink’.

Note: For signal panes without an X-axis bar (i.e., all but the bottom-most signal), the zoomed data are displayed independently of any X-axis, and the time synchronization between signals gets lost. Click the ‘Auto-scale All’ button to restore synchrony.

§ Axis Scroll Bars

Figure 4‑41. Axis Scroll Bar

The X-axis scroll bar is directly underneath the X-axis, while the Y-axis scroll bar is on the far right-edge of the Scope window. The amount of applied magnification is reflected in the size of the X- and Y-axis scroll bar slider buttons. Click and drag the offset bar slider button, or use their outer directional buttons to move the displayed signals in the desired direction. The Y-axis scroll bar controls the active signal pane.

§ Center:

Figure 4‑42. Center Button

Center the active signal. If the data are displayed off-center (or off-screen), the Center button repositions the active sweep so that the mean of the data is centered in the signal pane. The Y-axis offset is automatically adjusted, while the Y-axis scaling is kept unchanged.

§ Autoscale All Axes

Figure 4‑43. Autoscale All Axes

Autoscale all visible signals. This button is a convenient way to return all X-axes to their full sweep duration and all Y-axes to their visible sweeps data limits.

§ Amplitude Meters

Amplitude meters are displayed on the right border of signal panes for physical (non-virtual) channels. They provide visual feedback on the integrity of your data recordings, similarly to how audiometers monitor audio signals.

Figure 4‑44. Amplitude Meters

For the Triggered Sweeps acquisition mode, each displayed signal has its own Y-axis amplitude meter on the inner right side of the associated Scope pane.

For the Continuous Sweep acquisition mode, or if acquisition has not yet started, these meters are completely black.

The height of the colored meter bars represents a Signal’s data range vs. the full recording range of the IPA digitizer. The color of the meter bar corresponds to the data “health”:

· Green Good: Signal within appropriate range

When the recorded data are within proper amplitude limits, the amplitude meter is green.

· Yellow Caution: Signal approaching limits

(within 10% of range limits)

When there is a danger that saturation will occur, and the data are near saturation levels, the amplitude meter is yellow, as a warning sign to decrease your hardware gain. In this case, consider reducing the hardware gain.

If too little hardware gain is applied, and the recorded signal has insufficient amplitude resolution, the amplitude meter will be a thin yellow line. When this occurs, the digitizer is not utilizing enough bits to accurately represent the data, and your signal of interest may be contaminated with noise. In this case, consider increasing your hardware gain.

· Red Danger: Signal too large or small

(within 1% of range limits)

If too much hardware gain is applied, the recorded signal will be in danger of saturating, i.e. your data will exceed the amplitude limits of the digitizer. In this case, those data points are substituted with the maximum amplitudes of their input channel. When an amplitude meter is displayed in red, it indicates that the data might have gone out of range and might not be valid.

Other Buttons

	Clicking this button displays digitized analog data in the Scope window input signals. When you click the ‘Start Acquisition’ button, the Scope is cleared, data recording starts, and the button changes to ‘Stop Acquisition’. If measurement graphs are enabled, a “child” Analysis window is also displayed that plots sweep-by-sweep measurements. The Scope window updates every 200 ms. If no paradigm is running, an “Auto-triggered Paradigm” is generated and assigned a Paradigm name with the current Date/Time. If Metadata prompts are set for Routines or Paradigms, the Confirm Metadata Settings dialog displays just before recording occurs.
	Determines how data acquisition is terminated. When the ‘Stop Acquisition’ button is clicked, data acquisition is immediately halted, even if this occurs in the middle of a sweep. In this case, any partial sweep that was in progress is not saved with the data. However, if the ‘Stop at End of Sweep’ checkbox is enabled, then the current sweep will complete before data acquisition is stopped, and the last recorded sweep will be a complete sweep of data. If no sweep is currently in progress, acquisition will stop at the end of the next sweep to be recorded.
	Opens a special Real Time Measurements & Graphs dialog, where all edits apply instantly to the measurements and graphs, even during acquisition. These changes temporarily override the loaded routine for quick interactive analysis.
	Opens the Routine Editor dialog, for configuration, management and activation of Routines. Routines can be configured for: Data acquisition parameters Input channels Output channels Stimulus waveforms Analysis measurements
Layout	Paste all visible Scope signals and analyses into a Layout window. If no Layout window is open, a new one is created.
Persistence Display	When enabled, sweeps are not cleared from the display until a new acquisition is started. When disabled, all sweeps are cleared and only the next acquired sweep is displayed.
Sweep #:	The active sweep # vs. the total ‘Number of Sweeps’.

Table 4‑3: Other Buttons

Right-click Menus

Y-Axis

Autoscale All Axes Scale all signals X- and Y-Axes

Autoscale Axis Scale a signal’s Y-Axis, no change to the X-Axis

Full-scale Axis Set a signal’s Y-Axis to the full-scale range

Axis Properties Modify the axis style and components

Hide Signal <name> Remove the signal from the Scope

Show Signal <name> only Remove all other signals from the Scope

Stack All Signals Display all signals in a single column

X-Axis

Autoscale All Axes Scale all signals X- and Y-Axes

Full-scale Axis Set a signal’s X-Axis to the full-scale range

Axis Properties Modify the axis style and components

Window background

Autoscale All Axes Scale all signals X- and Y-Axes

Add Annotation Insert a text box into the window

Export Graphics Copy the data to a graph window

Colors graph background

all axes

all grids

all tick labels

all axis labels

Hide Signal <name> Remove the signal from the Scope

Show Signal <name> Only Remove all other signals from the Scope

Stack All Signals Display all signals in a single column

Data area

An Igor menu displays with numerous options to modify sweeps, such as marker symbols and lines.

Marquee

Click and drag the mouse to surround a region of interest:

Expand Magnify all axes to the marquee limits

Horiz Expand Magnify the X-axis to the marquee limits

Vert Expand Magnify the Y-axis to the marquee limits

Shrink Shrink the axes to the marquee size

Horiz Shrink Shrink the X-axis to the marquee size

Vert Shrink Shrink the Y-axis to the marquee size

Extract Template Copy the (last) sweep to the Template Editor

4.1.8 Solution Editor

Control perfusion systems with “named” solution control settings.

Figure 4‑45. Solution Editor

You can create a named list of solution control “valves” to manage your analog and digital commands to physical valves and solution changers.

Save Save the solution list as a ‘*.spo’ file.

Configure Opens the Configure Hardware Controls dialog to set solution types, channels, and outputs.

Solution Pool Files Drop-down list operations affect the entire “Solution Pool”.

New Solution Pool Create a blank Solution Pool.

Load Solution Pool Load the Solutions of a previously saved Solution Pool file into the Solution Pool.

Revert to Last Saved Undo any unsaved changes to the Solution Pool.

Save Solution Pool Save the Solution Pool using its existing file name and path.

Save Solution Pool As Save the Solution Pool to a new file, and switch to the new file.

Save Solution Pool Copy Save the Solution Pool to a new file, but do not switch to the new file.

Note: Default file names are auto-incremented from the previously loaded Solution Pool name.

The file path and file name of the loaded Solution Pool file is displayed.

[ Off, 1 – 16 ] Manually select a radio button to activate a solution configuration. Only one solution can be active at a time.

Double-click on a field to edit it, or click-and-drag to move it up or down in the table.

Concentration Enter a concentration value for the solution, including units, such as “0.5 nM”.

Access the concentration value from the last-used Solution Editor valve (for “Chemical Stimulant” solution types only) with the Special ID ‘Solutions’.

Description Your own text description of the solution.

Configure Hardware Controls

Close Dialog button

[ # Solutions ] Set the number of solution configurations (valves). Changing this number creates a new solution pool.

[ 2, 4, 8, 16 ]

[ Solution Type list ] A list of predefined solution types.

· Bath Solution

· Pipette Solution

· Chemical Stimulant

· Rinsing Solution

[ Output Channel ] Select a physical output channel and set its value.

· No Output

· AuxOUT1 [ ±10.000 V ] Analog output voltage

· AuxOUT2 [ ±10.000 V ] Analog output voltage

· DigOUTWord [ 0 – 255 ] Decimal value of an 8-bit digital word

· DigOUT1 – 8 A single digital bit is set “high”

4.1.9 Template Editor

Manipulate and manage templates for use in command waveforms.

Figure 4‑46. Template Editor

Template Pool Files

New Template Pool Create a blank Template Pool

Load Template Pool Load the Templates of a previously saved Template Pool

Revert to Last Saved Undo any unsaved changes to the Template Pool

Save Template Pool Save the Template Pool using its existing file name and path

Save Template Pool As Save the Template Pool to a new file, and switch to the new file. The default file name is the same as the original file name.

Save Template Pool Copy Save the Template Pool to a new file, but do not switch to the new file. The default file name increments.

Merge Template Pools Insert the Template from a previously saved Template Pool file into the loaded Template Pool

The file path and file name of the loaded Template Pool file is displayed.

Import Select a template file (*.ibw).

Alternatively, in a Scope window or preview pane, click and drag the mouse to surround a region of interest with a bounding box (the “marquee”). Right-click in the box and select ‘Extract Template’. A template with the signal name is added to the template list.

An extracted template is composed of a single sweep:

· Scope window (Acquisition): last sweep

· Scope window (Analysis): selected sweep

· Preview pane: last or selected sweep

Note: ‘Extract Template’ is not implemented for graphs or the Data Navigator preview pane. Also, it is only valid with monotonically increasing or decreasing X-axes.

Export Export the selected template to a separate file.

Rename Edit the name of the selected template. Allowable characters are A-Z, a-z, 0-9, and “_”. Special characters are not allowed; spaces are replaced by an underscore.

Duplicate Add a copy of the selected template to the list. The new template name’s number is appended or incremented.

Delete Remove the selected template from the list.

Revert Discard any unsaved changes to the selected template.

Save Pool Save the template pool using its existing file name.

To Clipboard Copy the template to the clipboard.

Convert Adjust the template sampling rate.

Smooth Apply smoothing to the template.

· Off

· Boxcar A fast time-domain filter with excellent 0 – 100% rise-time response

· Gaussian A standard filter with exellent 10 – 90% rise-time response

Factor Adjust the template scaling factor.

Offset Adjust the template offset.

Do It Apply the adjustments to the template parameters.

Template Names A list of the loaded templates.

Click on a Template entry to make it the active one.

Double-click on a Template Name to rename it.

Click-and-drag a Template entry to reposition it in the list.

Template Parameters Parameter settings description.

Show Preview Display the selected template in a preview pane.

The preview pane X- and Y-axes can be controlled in two ways:

· Hover the mouse over an axis line until the cursor turns into a double-headed arrow, then scroll up or down to contract/expand the axis.

· In the preview, click and drag the mouse to surround the region of interest with a bounding box (the “marquee”). Right-click in the box and select one of the expand/shrink options.

4.2 Analysis

Routines contain measurement settings that are run during online data analysis, and which can be re-applied during offline data analysis. Specialized analyses can also be applied to data via the Data Navigator.

For complete flexibility in controlling how analyses are performed, Paradigms can run virtually any SutterPatch command, Igor analysis or user-defined function. To execute such commands “per sweep”, use the paradigm Execute step in conjunction with ForEachSweep loops.

4.2.1 Action Potential Analysis

Action potentials (AP) are automatically analyzed with this dialog. Select a data signal in the Data Navigator, then click the Available Actions button (or right-click the signal).

Figure 4‑47. Action Potential Analysis

Sweep # The sweep number of the selected event.

When set to ‘0’, this indicates that averaged Results measurements are being displayed.

Event Time Timepoint of the selected event peak.

[Event pane] A graph of the selected event.

Event [ # of # ] Selected event number vs. total events.

Start Run the Action Potential Analysis.

Event Length (s) Event duration in the Event pane (also highlighted in red in the Sweep sub-window.)

Threshold (V) This voltage level needs to be reached or exceeded for analysis of an event to be triggered.

APD measure at: Set the Action Potential Duration percentile

This measures the duration of an event at an amplitude based on a percentile of the event repolarization amplitude (peak voltage - threshold potential).

APD at % repolarization

· 50%

· 80%

· 90%

· 100%

Analysis Start (s) Start time for data analysis in a sweep.

Analysis End (s) End time for data analysis in a sweep.

Average Display the measurement averages in the Results panel, and reset the ‘Sweep #’ to ‘0’.

Save Results Results are displayed in both a Layout window and a table - prior Results from the loaded data signal are overwritten.

Action Potential Analysis Results A Layout window report is created.

Total time analyzed Includes the Start/End times for all sweeps (s)

Number of events detected Total number of events found

Event Frequency ( Hz )

[ Event graph ]

[ Phase plot ] dV/dt (V/s) vs. mV

Results A table of all events results

[ ] Row number, one row per event

Sweep Number Sweep number the event is in

Event Time (s) Time point of the event start

Threshold (V) Amplitude of the event threshold

Threshold Time (s) Time point of the “trigger” threshold time

Peak (V) Amplitude of the event peak

Peak Time (s) Time point of the event peak

AP Duration (s) Duration of the action potential

AHP (V) Peak amplitude of after hyper-polarization

AHP Time (s) Time point of after hyper-polarization; the event re-crosses the threshold amplitude at this time.

Show/hide the sweep sub-window (below).

[ ] Displays a graph of the sweep containing the selected event.

Show/hide the results sub-window (on the right).

[ ] Displays a sub-window of action potential measurements

Figure 4‑48. Action Potential Measurements

[ Phase plot ] dV/dt (V/s) vs. mV

Threshold potential = Event starting amplitude (time from Threshold setting)

[ mV (µs) ]

This is the biological start of an event, which occurs when [signal slope > 1 mv/100 µs]; its timepoint is reported relative to the ‘Threshold’ setting timepoint.

Note: The exact ‘Threshold potential’ timepoint is based upon differentials using a central differences algorithm:

Peak = The largest excursion of the event (time from ‘Threshold’ setting)

[ mV (ms) ]

Time is reported relative to the ‘Threshold’ timepoint.

APD ( %) = Action Potential Duration of the event at ( %) repolarization

[ %, ms ]

AHP (min) = “After hyperpolarization” of the event (time from ‘Threshold’ setting)

[ mV (ms) ]

AHP is when the action potential repolarization phase drops to its lowest point below the resting membrane potential, i.e. during the hyperpolarized refractory period of the cell.

Events found = The total number of events found in the analyzed data

Event frequency = The average frequency of events found in the analyzed data

4.2.2 Analysis Editor

Various manipulations can be applied to your experiment analyses and graphs.

Figure 4‑49. Analysis Editor

Select Choose how to view the data:

Table View a spreadsheet of the Analysis data

Column 1: Row number

Column 2: X-data for the first graph

Column 3: Y-data for the first graph

Column m – n: Pairs of X- and Y-data columns repeat for each additional graph.

The first graph (X-Y-pair) that uses X-axis time units populates its X-data column with time values; subsequent graphs that use X-axis time units have blank X-data columns, as they use the first time-column created.

Note: The first data point is in row 0, so the last data point is in row [ N – 1 ].

Row N is a blank row that contains grayed-out cells. It is used to manually add extra rows of data to the table. Once a number is entered into one of these cells, the blank (gray) row is automatically moved to the row beneath it.

Warning! Editing the table will permanently alter the data.

Graph-[1 – 8 ] Select an Analysis graph for the selected data wave. The graph number refers to its original Analysis window position.

To Clipboard Copy the selected table or graph to the operating system (OS) clipboard.

To Layout Copy the selected table or graph to the Layout window.

Files Save or open an analysis graph file.

Export Graphs Save the entire graph as a multidimensional Igor Binary Wave (*.ibw) file

Export Graph X-column Save the X-column data, including labels, s a 1-D wave (*.ibw) file

Export Graph Y-column Save the Y-column data, including labels, as a 1-D wave (*.ibw) file

Import Graphs Open and display a saved graph

Note: There is no import of 1-D wave data

Options Show Axes Color Display a background color for the axes

Show Grid Display X & Y grid lines in the graph

Show Error Bars Display SEM error bars for averaged data

Show Markers Display data points with marker symbols

Show Lines Display a line between data points

(Toggle between ‘markers’ and ‘lines’, or both)

Cell Separator: TAB Use tab separators when exporting a table (e.g. to Excel)

Cell Separator: Comma Use comma separators when exporting a table (e.g. to MS-Works)

Include Column Labels Column labels appear on the first line of an exported table

Operations Duplicate Append a copy of the graph

Delete Graph or Table Delete a particular graph or an entire table

Delete Analysis Delete the entire analysis wave

Note: An analysis cannot be deleted if it exists in a graph or analysis window - it must first be closed.

Math Normalize: zero to maximum Rescale the largest point to 1.0

Normalize: minimum to maximum Rescale the smallest point to zero, and the largest point to 1.0

Invert Reverse the Y-axis sign of the data

Append Displays available analysis wave names for appending

Select an analysis wave for appending with the loaded wave. Time-course data data are plotted relative to the loaded analysis wave’s “time zero”.

Average Displays available analysis wave names for averaging

Select an analysis wave to be averaged with the loaded wave. A weighted average is performed, i.e., the number of data sets is accounted for when averaging in new data.

Two new entries are inserted into the wave list after the loaded wave:

1. The averaged wave

2. The SEM (Standard Error of Means) data points wave

If Options / Show Error Bars is enabled, the SEM data are used to display error bars in the corresponding averaged-data graph.

Standard Error of the Mean (SEM) Algorithm

SEM = √ ( SumSq – Mean² * N ) / (N-1)

SumSq = sum of all squared samples

Mean = sum of all samples / N

Note: The SEM algorithm is similar to the Standard Deviation

“√ ( variance )”, but using ‘Mean’ vs. ’sum of all samples’.

Scale and add List of available analysis wave names for scaling.

Use to combine waves, with optional scaling and offset applied.

When the Factor is ‘1.00’ and the Offset is ‘0.00’, this operation will simply add the selected wave to the displayed wave.

Factor Set a scaling factor for the selected wave to be added. To subtract a wave, change the Factor to a negative number, such as ‘-1.00’.

Offset Set an offset for the selected wave to be added.

Show Cursor Info Open the Cursor bar to see X & Y data values, and to set a fitting range.

A: pnt: The fit beginning data-point symbol, and its point number field.

B: pnt: The fit ending data-point symbol, and its point number field.

1. Select symbol A and drag it onto the beginning data point (or enter the data-point number in the ‘pnt’ field.)

2. Select symbol B and drag it onto the ending data point (or enter the data-point number in the ‘pnt’ field.)

3. The data points X- and Y-coordinates are displayed.

4. Right-click in the graph and select ‘Quick Fit’ for a list of built-in Igor fitting functions.

5. The fit is displayed in the graph, and the fitting information is written to the Log and Command windows.

Options menu

· One Mover Moves All

· All Styles Change the symbol graphics

· Show Cursor Pairs Display up to 5 sets of cursor symbol pairs

After the cursor endpoints are set, right-click in the graph and select ‘Quick Fit’ for a list of built-in Igor fitting functions to apply to the cursor range. The fit will be displayed in the graph, and the fitting information is written to the Log and Command windows.

Analysis Wave Names Loaded analyses available for manipulation.

Analysis Parameters

· Series: R#_ name of the Series

· Graphs: # number of graphs in the analysis wave

· Points: # number of data points in the graph

· Start: # start time of analysis wave

· Average: # number of graphs averaged or appended

Tip: If the Analysis Parameters text is not fully visible, increase the width of the Analysis Editor window.

[Graph & Table pane] Data point markers are plotted, or a numeric table is displayed.

X- and Y-axes can be magnified to be larger or smaller. Place the mouse cursor in the axis ticks region, then scroll the mouse wheel up or down. The axis ticks region does not include the tick label (numbers) area.

4.2.3 Analysis Window

Scope measurements are plotted in an Analysis window docked on the right side of the Scope window. An Analysis window can be resized or closed, but not undocked from the Scope window.

Figure 4‑50. Analysis Window

Analysis measurements are configured in the Routine Editor Real Time Measurements & Graphs section. A separate pane is created in the Analysis window for each enabled Measurement graph.

Online measurements are plotted during data acquisition in real-time.

Data can be selected for offline review or analysis via the Data Navigator. When stored data are rerun for analysis, the data displays in a Scope window, and the analyses are graphed in an accompanying Analysis window. The last measurements applied to the data are automatically used to reanalyze the data.

Save Analysis This button saves the displayed analyses with the Experiment.

Saved analyses are viewable in the Analysis Editor or from the Data / Data Browser: Data / Analysis folder.

[Graph pane] X- and Y-axes can be magnified to be larger or smaller. Place the mouse cursor in the axis ticks region, then scroll the mouse wheel up or down.

Note: The axis ticks region does not include the tick labels (numbers).

Symbols

Symbols can be manually overlaid onto Analysis window data points.

1. Select the Analysis window and click CTRL-I, to display the symbols tool bar.

2. Select symbol A or B.

3. Drag it onto a data point, or enter the data point number in the ‘pnt’ field.

4. The data point X- and Y-coordinates are displayed. When two symbols are placed into the graph, the delta X-Y value is also displayed.

‘Options’ menu

· One Mover Moves All Move all symbols together with a control

· All Styles Change the symbol graphics

· Show Cursor Pairs Display up to 5 sets of cursor (symbol) pairs

Note: If the Analysis window is closed when a Layout graph is created, Analysis graphs will not be included in the Layout graph.

4.2.4 Data Browser

The Data Browser displays all of the Experiment’s data objects, such as data waves, analysis graphs, layouts, images, metadata, Paradigms and Routines.

Figure 4‑51. Data Browser

Objects are displayed in a tree structure, using a path “root” of ‘SutterPatch’.

Recorded data are listed in the ‘Data’ subfolder, arranged per Signal.

Right-click Menu

Display Display the Analysis data in a visual graph.

Edit Display the Analysis data in a numerical table.

SutterPatch signal data are stored in two-dimensional data waves, with one column per trace, and one row per sample point.

Warning! Editing data here will permanently alter the raw data. Modify at your own risk!

Copy Full Path Copy the object’s path to the clipboard. This refers to Igor’s internal data folder in the Data Browser, not the computer OS file system. This path can be used by advanced Igor users in user functions and executable commands.

4.2.5 Data Navigator

The Data Navigator window organizes and displays all levels of data and controls for the loaded Experiment.

Figure 4‑52: Data Navigator

Data tree hierarchy The hierarchy of sorting levels is displayed in this pane.

Build Hierarchy Re-organize the data tree using custom settings.

Select parameter group Organize by metadata parameters:

(availability depends on Preferences)

All Categories

Frequently Used

Experiment Hierarchy

Tag

Operator

Preparation - Animal

Preparation - Tissue

Preparation - Cell

Experiment

Amplifier

Data Acquisition Settings

Imaging

Stimulus

Available parameter Click on a specific parameter to select it.

Click on the “copy” button to insert the selected parameter into the data hierarchy.

Hierarchy The Hierarchy pane displays the new sorting hierarchy. The entries can be re-organized by selecting an entry and clicking on the Up/Down keys to reposition them, or using the ‘Del’ key to remove them.

Up and Down positioning keys.

Clear Hierarchy Remove the existing hierarchy. The current Experiment’s raw data are organized in a simple time sequence.

Default Hierarchy Restore the default experimental hierarchy: Experiment > Paradigm > Routine > Series > Sweeps

Do It Click on the ‘Do It’ button to apply these changes to the data tree.

Data Tree Window The current Experiment’s data are arranged in the data tree (down to the Sweep level.)

Expand All nodes of the data tree are expanded down to the Signal level.

Collapse All All nodes of the data tree are collapsed up to the Paradigm level.

Last Series The last Routine’s first signal is highlighted in the data tree and its data are displayed in the Preview pane below it.

Import Select a previously saved SutterPatch experiment to incorporate into the current experiment.

Analyze The selected Routine or Series signals are opened in the Scope window for analysis.

Review The selected Paradigm’s signals and data are opened in the Scope window for review.

Note: Depending upon the data level, the same button displays the Analyze or Review commands.

Show Preview / Hide Preview The display is based upon the data level

Note: The Preview pane does not support mouse operations.

Paradigm: Metadata Wave Name: Click to display the Paradigm’s metadata

Images: Open any saved images

[ Preview sub-pane ] Displays the first signal of the first Routine

Routine: Metadata Wave Name: Click to display the Routine metadata

Signals: Number of signals

Sweeps: Number of sweeps

Routine Data Name: Click to display the Routine parameters

Analysis: Open any saved analyses into the Analysis Editor

Images: Open any saved images

[ Preview sub-pane ] Displays the first signal of the selected Routine

Signal: [ Preview ] Displays the selected Signal

Sweep: [ Preview ] Displays the selected Sweep

Available Actions button Actions are based upon the data level

(or right-click menu)

Paradigm: Review Display the Series data in the

Paradigm Review window

View Metadata Display the metadata in the Metadata Review window

Discard Paradigm Data Delete the selected Paradigms and their Series and remove from the Experiment

Routine: Analyze Display the Series data in a

Scope (reanalysis) window.

View Metadata Display the metadata in the Metadata Review sub-window

View Routine Display the Routine parameters in the Routine Review window

Show Data in Data Browser Open Igor’s Data Browser window.

Discard Series Delete the selected Routines and their Series, and remove from the Experiment

Export as ATF File Save the routine formatted as an “Axon Text File” (*.atf)

Signal: Analyze Display the Series data in a

Scope (reanalysis) window. All signals and sweeps display

Event Detection Open to analyze synaptic events (minis, etc.)

Action Potential Analysis Open to analyze action potentials

Edit Display the Series signals as numeric columns in an editable table

Average Signal Average all sweeps in the signal and display in the Analysis Editor

View Metadata Display the Series metadata in the Metadata Review sub-window

Copy Full Path Copy the Series internal Igor path to the clipboard.

( root:SutterPatch:Data:Series_name )

Show Data in Data Browser Open Igor’s Data Browser window

Sweep: Analyze Display the Series data in a

Scope (reanalysis) window

Edit Display the Series data as numeric columns in a table

Extract Sweep Create a graph of the sweep in the Analysis Editor.

Show Data in Data Browser Open Igor’s Data Browser window

4.2.6 Data Table

The Data Table provides direct access to the sample points in a data Series, using a spreadsheet-style presentation.

Figure 4‑53. Data Table

Data Tables are accessed from the Data Navigator by highlighting a data Series, right-clicking on it, and selecting the ‘Edit’ command. Alternatively, use the menu item Data / Data Browser and select a Series from the Data folder; then use the right-click menu item ‘Display’.

To allow adding data to the table, the last row of data in the table is followed by a final row of blank (gray) cells. Manually entering data into the final blank row causes a new last row of data to be created in the table, followed by a new final blank row.

4.2.7 Equation Editor

The Equation Editor manages simple or complex expressions that evaluate to a value. Such math equations can be used to create stimulus waveforms, and for data analysis.

Access the Equation Editor from the SutterPatch menu.

Figure 4‑54. Equation Editor

Equation Pool Files Equations are created and saved in an Equation Pool file

New Equation Pool Create a blank Equation Pool file

Load Equation Pool Load the Equations of a previously saved Equation Pool file into the Equation Pool

Revert to Last Saved Undo any unsaved changes to the Equation Pool

Save Equation Pool Save the Equation Pool using its existing file name and path

Save Equation Pool As… Save the Equation Pool to a new file, and switch to the new file. The default file name is the original file name.

Save Equation Pool Copy… Save the Equation Pool to a new file, but do not switch to the new file. The default file name has ‘Copy of’ prepended to it.

Merge Equation Pools Insert the Equation from a previously saved Equation Pool file into the loaded Equation Pool

Note: Equation Pool files are simple text files that can be directly edited.

New Create a blank Equation

Duplicate Add a copy of the selected Equation to the Equation Pool

Delete Remove the selected Equation from the Equation Pool

Revert Select an Equation and click the Revert button. All editable steps are reset to their last saved settings.

Save Pool Save the Equation Pool using its existing file name

Edit Make edits to the ‘Equation’ field

Check Equation The equation is evaluated (for sweep #1), and if valid, it reports that the syntax is “ok”.

Label Column of editable equation names, for quick usage in place of the equation.

Equation Column of equations in free-form text fields

Insert special identifier

The following special identifiers can be used as special functions in equations:

loop (active paradigm ForLoop count)

sweep (active paradigm EachSweep count)

LastSweep (active paradigm sweep count of last sweep)

Processing can occur before or after the last sweep of a series.

Example: In a Paradigm ‘If’ step, compare ‘sweep’ numbers in a ForEachSweep loop.

ForEachSweep

EachSweep, Target=IV

If, Left=sweep, Operation= ‘=’, Right=LastSweep-1

Alert, Text=LastSweep, DoBeep=true

EndIf

ForEachEnd

AqStopped (last acquisition was stopped)

The last Routine-Series did not complete by itself.

ParadigmTime (time at start of paradigm, s)

RoutineTime (time at start of routine, s)

Stimulant (last applied stimulant concentration)

From the Solution Editor ‘Concentration’ setting for a solution configured as a ‘Chemical Stimulant’.

time (present date-time, s)

timer (timer time, s)

m[1..16] (n’th analysis measurement value)

gx[1..8] (n’th analysis graph x value)

gy[1..8] (n’th analysis graph y value)

r[1..16] (n’th routine stimulus variable)

p[1..16] (n’th paradigm variable)

Hold[1..8] (holding of n’th output channel)

Input (Input variable on paradigm window)

AuxIN[1..8] (reading of auxiliary input, V)

A single-point voltage reading from an Auxiliary Input channel of the IPA system, such as from a slowly changing temperature probe.

Note: This usage does not require setting up a Routine Input Channel.

Imon (amplifier current reading, A)

In the Amplifier Control Panel (pA).

Vmon (amplifier voltage reading, V)

In the Amplifier Control Panel (mV).

Mean[name or count, start,width] (mean of given input signal)

ActiveProbe (active probe)

NumProbes (number of probes)

CCMode (amplifier current clamp)

VCMode (amplifier voltage clamp)

Hold (IHold in CC-mode, VHold in VC-mode)

[ ±0.000,000,020 A (±20,000 pA), or ±1.000 V (±1000 mV) ]

IHold (amplifer holding current, A)

[ ± 0.000,000,020 (±20,000 pA) ]

IHoldOn (amplifier holding current On)

VHold (amplifier holding voltage, V)

[ ±1.000 V (±1000 mV) ]

VHoldOn (amplifier holding voltage On)

IGain (amplifier current gain, V/A)

The gain of the active voltage-clamp ‘Current’ input channel.

· 0.5 mV/pA

· 1 mV/pA

· 2.5 mV/pA

· 5 mV/pA

· 10 mV/pA

· 25 mV/pA

VGain (amplifier voltage gain, V/V)

The gain of the active current-clamp ‘Voltage’ input channel.

· 10 mV/mV

· 20 mV/mV

· 50 mV/mV

· 100 mV/mV

· 200 mV/mV

· 500 mV/mV

Filter (amplifier input filter, Hz)

Apply a filter to the input channels.

Use a preset value, or a 10% threshold between the preset values is applied to the equation (to avoid over filtering).

· 500 (500 Hz)

· 1000 (1 kHz)

· 2000 (2 kHz)

· 5000 (5 kHz)

· 10000 (10 kHz)

· 20000 (20 kHz)

Offset (amplifier pipette offset, V)

OffsetLock (amplifier pipette offset lock)

VTrack (amplifier tracking potential, V)

VTrackOn (amplifier tracking potential On)

ECompMag (amplifier electrode compensation magnitude, F)

ECompPhase (amplifier electrode compensation phase, fraction)

ECompOn (amplifier electrode compensation On in CC mode)

CmComp (amplifier cell compensation Cm, F)

RsComp (amplifier cell compensation Rs, Ohm)

RsCompOn (amplifier cell compensation Rs On)

RsLag (amplifier Rs correction lag, s)

RsPred (amplifier Rs prediction, fraction)

RsCorr (amplifier Rs correction, fraction)

RsCorrOn (amplifier Rs correction On)

Bridge (amplifier bridge balance, Ohm)

--------

Relectr (electrode/seal/access resistance, Ohm)

Rmemb (membrane resistance (Cell mode), Ohm)

Cmemb (membrane capacitance (Cell mode), F)

Other identifiers are forwarded to Igor Pro's ‘Execute’ command.

Equation Extras

Constants

true 1

false 0

The following constants have 27-digit precision:

e 2.71... (Euler’s number)

pi 3.14... (π)

Parsing and Operators

Equation parsing is executed from left to right, processing the highest precedence operators first:

Precedence	Type	Operator
8	Comment	;
7	Exponentiation	^
6	Logical operations: Unary Negation, Logical Negation	-, !
5	Multiplication, Division, Remainder	*, /, %
4	Addition, Subtraction	+, -
3	Bitwise comparison: And, Or, Nor, Xor	&, \|, nor, xor
2	Logical operations: And, Or, Conditional If	&&, \|\|, If( ? : )
1	Relational operations	>, >=, <, <=, !=
0	All other operations	Round, trunc, ceil, floor, exp, sqrt, ln, log, sin, cos, tan, asin, acos, atan, abs, rad, deg, noise, random

Table 4‑4: Equation Parser

Note: In the SutterPatch equation parser, Relational operators (Level 1) use standard math priority, i.e., their precedence is lower than Logical operators (Level 2), while in the Igor Pro parser, Relational operators have higher precedence than Logical operators. Given these conflicting priorities, it is advisable to use parentheses to define these operations’ priorities.

All characters to the left of a Comment semi-colon symbol are ignored by the SutterPatch parser; however, the Igor Pro parser uses semi-colons to separate multiple commands on the same command line.

The ‘Conditional’ operator “If( ? : )” is evaluated as:

If( statement ? Result-when-True : Result-when-False )

Note: the " : " is actually a colon with 2 blank spaces around it.

Example: Set a threshold (lower limit) on cursor measurement values

~~Example:~~ s~~et a threshold~~ ~~(lower~~ ~~limit~~) ~~on cursor measurement values~~

If( m[1] > 2p ? m[1] : NaN )

While the bitwise left and right shift operators in Igor Pro 7 are not supported in SutterPatch, they can be constructed from existing operators:

Example: shift left by “n” bits

* 2^n

Example: shift right by “n” bits

/ 2^n

Syntax

All equations use the same syntax as Igor Pro 7, with a few additions:

Three kinds of brackets [ ], { }, ( ), can be used equivalently to improve the clarity of nested expressions.

Numeric values can be written in scientific E-notation using exponents:

5e-12 (5 picoamps)

or in equivalent engineering notation using unit prefixes:

5p (5 picoamps)

Prefix	Exponent	Prefix Name	Prefix	Exponent	Prefix Name
k	10³	Kilo	m	10^-3	milli
M	10⁶	Mega	µ (or u)	10^-6	micro
G	10⁹	Giga	n	10^-9	nano
T	10¹²	Tera	p	10^-12	pico
P	10¹⁵	Peta	f	10^-15	femto
E	10¹⁸	Exa	a	10^-18	atto
Z	10²¹	Zetta	z	10^-21	zepto
Y	10²⁴	Yotta	y	10^-24	yokto

Table 4‑5: Engineering Notation

Insert an equation from the Equation Editor Pool into an Equation field by entering “#” followed by the label of the equation, e.g. “#MyLabel(5)”. This passes the argument “5” to the equation labeled “MyLabel” for evaluation.

Example: Using an LED light source

To stimulate in increments of light intensity, use an equation to transform light intensity values in Routine variables into actual stimulus values with amplitudes in volts.

Build an equation in the equation pool as follows:

equation = ln( r[1] ) * 2.55 + 3

The natural log of the Routine Variable r[1] is multiplied by 2.55 and added to 3.

label = power_to_volts

In the Waveform Editor, set a Segment Amplitude field to ‘Equation’, and enter the equation as “#power_to_volts”.

A simplified version of the Equation Editor allows Equations (and equation labels) to be used in the following areas:

Paradigm Editor

Steps: Amplifier, Checkbox, Set Variable, Sound, Write Log, If, Else If

Routine Editor

Input Channels: Virtual Channels: Math Type

Output Channels: Leak Hold, Waveform Editor: Amplitude, Duration

Measurements: Time to Threshold, X-Axis, Y-Axis

4.2.8 Event Detection

Post-synaptic currents and potentials, such as spontaneous and miniature EPSCs and EPSPs, are automatically analyzed by this application module. It uses an innovative deconvolution algorithm to find events with high temporal fidelity, while also improving the signal-to-noise ratio (SNR). Using this new technique, overlapping events are often resolvable.

Sweep # The sweep number of the displayed data.

Event Time The time of the event’s threshold crossing (s)

Amplitude The amplitude averaged around the peak by ±2 ms

concatenate sweeps Combine all sweeps into a single pseudo-sweep before processing

display template Display the ideal event’s template on top of the selected event in the graph - its Y-axis displays on the right edge of the graph.

Tip: To match the template to the data, hover the mouse cursor over the right Y-axis, and use the mouse wheel to rescale the template.

Threshold (xSD) A detection threshold representing the “Event Strength”, where a lower (“weaker”) number finds more events, while a higher (“stronger”) number finds less events. Adjust this threshold based on empirical testing of your data.

Lower # = more noise-based events (false-positives)

Higher # = more missed events (false negatives)

Note: The default threshold is set to 4 times the standard deviation of a Gaussian fit to an all-points histogram of the (Fourier) deconvolved data signal, which corresponds to ~0.0032 % false positive sample points.

Ampl Threshold Set an amplitude threshold for the minimum size of events

Analysis Start (s) Sweep time to start looking for an event threshold.

Analysis End (s) Sweep time to stop looking for an event threshold.

Event The current event number vs. total number of events

Remove Delete the current event from the analysis

Template Open the Template sub-panel to configure a template

Start Run the Event Detection analysis

Average The averaged event displays in the graph

Threshold Analysis A scatter plot of the Event Strength vs. Current is displayed in a sub-panel.

Save Results Event Detection Results A Layout window report is created

Total time analyzed = Includes the Start/End times for all sweeps (s)

Number of events detected = Total number of events found

Event Frequency = (Hz)

Average Event Amplitude = ±2 ms peak average (pA)

Standard Deviation of Event Amplitude = (pA)

Graphs: Cumulative probability vs. Amplitude

Amplitude (Average) vs. Time

Frequency vs. Sweep Number

Amplitude vs. Sweep Number

Results Table A table of columns is created

[ blank ] Row number with one row per Event

SweepNumber The sweep number the Event is in

SweepTime ‘Time to event’ from the start of sweep (s)

EventStrength A measure of how well the signal matches the template. (Lower is weaker, higher is stronger.)

EventAmplitude The Event peak amplitude (pA) ± 2 ms average

Template Create a template of a typical event as a double-exponential curve. The data will be deconvolved to this template for further analysis.

Event Polarity 1 = positive

-1 = negative

Rise Time (µs) Time constant (τ) for the rising phase of the template event

Decay time (µs) Time constant (τ) for the falling phase of the template event

Create Template Click this button to create the template

References

Deconvolution papers:

Pernía-Andrade AJ, Goswami SP, Stickler Y, Fröbe U, Schlögl A, Jonas P. A Deconvolution-Based Method with High Sensitivity and Temporal Resolution for Detection of Spontaneous Synaptic Currents In Vitro and In Vivo. Biophys J. 2012 Oct;103(7):1429–39.

4.2.9 Igor Analyses

Curve fitting options are located in the Analysis main menu:

· Curve Fitting Create your own fitting equation

· Quick Fit Use a pre-defined equation:

line

poly

poly_XOffset

gauss

Ior

exp_XOffset

dblexp_XOffset

exp

dblexp

sin

HillEquation

Sigmoid

Power

LogNormal

poly2D

Gauss2D

FitBetweenCursors

Weight from Error Bar Wave

Textbox Preferences

Example: Perform a fit on a section of a sweep:

1. In the Data Navigator, select the sweep, and use the ‘Extract Sweep’ command on it.

2. In the Analysis Editor, click the ‘To Layout’ button.

3. In the Layout window, right-click for the “Show Window” command.

4. With the Graph window active, select the main menu item Graph > Show Info.

5. In the Graph window cursor pane, drag cursors ‘A’ and ‘B’ onto the data.

6. Select main menu Analysis / Quick Fit and the fit of your choice.

Built-in Igor analyses are documented in the Igor Pro Help:

Fourier Transforms

Convolve

Correlate

Differentiate

Integrate

Smooth

Interpolate

Filter

Resample

Sort

Histogram

Compose Expression

Packages

Average Waves

Batch Curve Fitting

Function Grapher

Global Fit

Median XY Smoothing

MultiPeak Fitting

Percentiles and Box Plot

Wave Arithmetic

4.2.10 Layout Window

The Layout window is used to prepare your data for publication. Scope window input signals and associated Analysis window graphs are displayed in a Layout window for graphical arrangement and editing.

Layout windows can be manually created from a Scope window ‘Layout’ button

Layout windows can also be automatically created by a Paradigm running an Export step.

Note: The Layout window is often created behind other windows, and if blank, needs to be clicked on to update.

When a Layout window is open, the main menu “Layout” item displays. The ‘Append to Layout’ command allows you to append any graph object in the Experiment into the active Layout window.

Once a Layout window is created and filled, additional graphs are automatically appended to additional pages. The default arrangement is “2 x 4” graphs per page.

New Layout windows allow page layout configuration for the number of graphs per page (‘column’ x ‘row’). Control this setting in Preferences / Export Graphics or the Paradigm ‘Export’ step.

· 1 single pane

· 2 stacked panes

· 3 stacked panes

· 2 x 2 matrix

· 2 x 3 matrix

· 2 x 4 matrix

A toolbar displays in the upper-right corner of the Layout window. There are two configuration buttons at the top, which display different sets of buttons:

· Operate Mode Selection tools and object insertion

· Draw Mode Drawing tools

When you close a Layout window, you can choose to save it as a “macro”. To restore a Layout window, go to the Igor Pro menu Windows / Layout Macros and select the macro name. Refer to the Igor Pro documentation for further details about macros.

4.2.11 Metadata Review

Metadata describes the system environment (computer and amplifier) and user settings of Paradigms and Routines.

Metadata parameters are displayed via:

Data Navigator

· Select a Paradigm or Routine, and then its Metadata Wave Name in the Preview pane.

· Select a Paradigm or Routine, then click the 'Available actions' button and View Metadata.

· Right-click a Paradigm or Routine and select View Metadata.

Scope (Reanalysis) Use the View Metadata button in this window.

Note: User-defined parameters from all levels of the Set Preferences / Metadata / ‘Metadata detail level’ are displayed along with the automatic metadata.

Paradigm Data Displays the name of the Paradigm

Routine Data Displays the name of the Routine

· By Event System settings, routine settings, and defined metadata events are displayed per Signal. Highlighted fields are editable.

· By Parameter Includes an expanded set of software parameters displayed per category

Expand All All parameter settings are displayed

Collapse All All parameter settings are hidden

Automatic Metadata Parameters “By Parameter”

Tag

Tag Number

Tag Creation Timestamp

Timer Time at Tag Creation

Tag Signals

Tag Source Event

Operator

Experiment

Experiment Timestamp

Amplifier

Amplifier Sequence Number

Amplifier Manufacturer

Amplifier Model

Amplifier Serial Number

Amplifier Channel

Number of Available Headstages

Headstage Sequence Number (not displayed for a 1 headstage system)

Headstage Model

Headstage Serial Number (not displayed in Demo mode)

Instrumentation and Software

Interface Sequence Number

Interface Manufacturer

Interface Model

Interface Serial Number

Interface Input Channel (physical)

Interface Out. Ch. (physical or logical)

Interface Signal Type

Interface Number of Digital Outputs

Computer Name

Operating System Platform

Operating System

Software Environment

Software Environment Version

Software Environment Build

Software Environment Kind

Software Environment Serial Number

Data Acquisition Software

Data Acquisition Software Version

Data Acquisition Software Build

Paradigm

Paradigm Data Sequence Number

Paradigm Data Base Name

Paradigm Name

Paradigm Data Start Timestamp

Series (= Routine Data)

Series Sequence Number

Series Base Name

Routine Name

Routine Acquisition Mode

Routine Data Start Timestamp

Routn. Completed / Terminated Early

Number of Input Signals

Sweep

Sweep Number

Data Acquisition Settings

Active Headstage

Recording Mode

Current Gain

Voltage Gain

Headstage Gain

Headstage Feedback Mode

Filter Cutoff Frequency

Filter Type

Voltage Clamp Offset

Voltage Clamp Offset Lock On/Off

Current Clamp Offset

Current Clamp Offset Lock On/Off

Input Signal Name

Input Signal Units

Input Scaling Factor

Virtual Signal Scaling Offset

Input Full-scale Minimum

Input Full-scale Maximum

Virtual Signal Math Type

Virtual Signal Equation

Virtual Signal Source Channel

Virtual Signal Smoothing Factor

Electrode Fast Magnitude

Electrode Fast Time Constant

Whole-cell Compensation On/Off

Series Resistance Value

Membrane Capacitance

Series Resistance Prediction Value

Series Resistance Correction On/Off

Series Resistance Correction Value

Series Resistance Corr. Lag Time

Capacitance Neutralization On/Off (only displayed in CC mode)

Capacitance Neutralization Magnitude (only displayed in CC mode)

Capacitance Neutralization Phase (only displayed in CC mode)

Bridge Balance Resistance

Current Clamp Slow Tracking On/Off (only displayed in CC mode)

Current Clamp Slow Tracking Potential (only displayed in CC mode)

Command Signal Name 1

Command Signal Units 1

Command Full-scale Minimum 1

Command Full-scale Maximum 1

Command Holding Enabled 1

Command Holding Value 1 (if disabled, equals zero)

Command Signal Name 2 (not displayed for a 1 headstage system)

Command Signal Units 2 ( “ “ )

Command Full-scale Minimum 2 ( “ “ )

Command Full-scale Maximum 2 ( “ “ )

Command Holding Enabled 2 ( “ “ )

Command Holding Value 2 (if disabled, equals zero)

Digital Holding Pattern (1 à 8 or 16)

Amplifier Seal Test On/Off

4.2.12 Paradigm Review

The ‘Paradigm Review’ Scope window displays a time-course of all data recorded with the Paradigm, including all executed Routines and their Series data.

This view also displays the tags that occur between Routines.

See the Scope Window for usage of the window controls.

4.2.13 Reanalysis Measurements & Graphs

The Scope (Analysis) window Measurements button opens the ‘Reanalysis Measurements & Graphs panel. Use it to apply different analysis scenarios to recorded data by setting up or editing measurements on input channels and configuring analysis graphs.

SNAGHTML63904ae

Figure 4‑55. Reanalysis Measurements & Graphs

This dialog operates similarly to the Routine Editor: Real Time Measurements & Graphs dialog, with a few additions:

Measurement parameters from Select which Routine settings to record with

· Last settings (newly modified version)

· Routine Copy (last “executed” version)

· Routine Copy (original loaded version)

Edit Virtual Signals A virtual signal dialog allows modification of these “pseudo” input signals.

Figure 4‑56: Edit Virtual Signals

Virtual signals support a wide variety of data transformations. To enable a virtual signal, highlight a signal name, then click the ‘Do It’ button. Changes to the highlighted signal are saved when you click ‘Do It’, and changes in unhighlighted signals are discarded.

Input Unit The base unit of measurement from its Source signal. The resolution of the unit is automatically adjusted in the signal.

Scaling

Offset Apply an amplitude offset to the input signal (after any scaling)

For “mV” units, append with ‘m’ or ‘e-3’

For “pA” units, append with ‘p’ or ‘e-12’

Example: 5 picoamps using engineering notation: 5p

or in equivalent scientific E-notation: 5e-12

Factor Apply scaling to interpret the input signal data. Specify as a numeric value or an equation.

Note: The IPA data acquisition system uses a high-resolution 16-bit ADC with 64-bit data, so data resolution is not an issue when scaling input signals.

Math Type Apply a data transformation to a virtual input signal.

· Leak Leak subtraction

Math Equation: [Leak( Current; leak_function; 2)] ( a read-only field )

Source Signal Select an input signal to transform

Show averaged leak Display the average of the leak sub pulses

Leak zero segment Select a data segment without leakage, i.e. at "holding"

· Smooth Data filtering

Math Equation: [ Smooth( Current; Gaussian; 2 ) ]

( a read-only field )

Source Signal Select an input signal to smooth

Smoothing type

Gausian Smooth operations [ 1 – 32767 ]

# of smoothing operations to perform

Boxcar Smooth repetitions [ 1 – 32767 ]

# of smoothing repetitions to perform

Boxcar window points [1 – 99]

(odd values only)

# of points in boxcar “window”

· LineFreq AC line frequency reduction

Math Equation: [ LineFreq( Current; 60Hz) ]

( a read-only field )

Source Signal Select an input signal for noise reduction

Line Frequency 50 Hz

60 Hz

· Equation Specify a math equation for the virtual signal

(See the Equation Editor for more details.)

Math Equation: [ Equation( ) ]

Note: The complete equation is always available as a tool tip -hover the mouse cursor over the field.

“Specify math equation for virtual signal” dialog

[ Equation field ] A free-form text field

< Syntax status message >

Check Equation Check the equation syntax (for sweep #1). The equation is evaluated, and if valid, it reports “Syntax is “ok”.

Insert special identifier t[#] n’th input trace

The trace wave of the channel in Scope Position "n" is accessed using this substitute for the data folder path.

The simplest way to make a duplicate of a trace is by using a virtual signal of math type "equation" with the equation "t[1]" for the first Scope signal.

p[#] n’th paradigm variable

Undo All changes in the equation editing session are discarded

(See the Equation Editor for more details.)

· Stimulus Command Waveform

Math Equation: [ Stimulus( Current ) ] ( a read-only field )

Source channel Select an input channel – the waveform from its Parent Out Chan is used

· Differentiate Data transformation using differentiation

Source Signal Select an input signal to differentiate

· Integrate Data transformation using integration

Source Signal Select an input signal to integrate

Analysis Examples

Example 1: Plot the mean of the data (using sample routine IV)

1. Set measurement m[5] to the ‘Mean’ analysis and select signal Current1.

2. Enable graph [g5].

3. From the graph’s Y-Axis list, select m[5]. The Equation field displays:

m[5]

4. Set ‘X-Axis’ to ‘time’ .

5. Run the analysis.

6. An Analysis window displays a graph of the mean vs. time.

Example 2: Plot the difference between two measurements

1. Set measurement m[5] to the ‘Mean’ analysis and select signal Current1.

2. Set measurement m[6] to the ‘Mean’ analysis, using the same signal.

3. Adjust the m[6] cursors Start/End times so they do not overlap with the m[5] cursors.

4. Enable graph [g6].

5. For the graph’s Y-Axis, select ‘Y-Equation’ and enter the equation as:

m[5] – m[6]

6. Set the X-Axis to ‘time’.

7. Run the analysis.

8. An Analysis window displays a graph of the difference vs. time.

4.2.14 Routine Review

The Routine Review window is similar to the Routine Editor Routine Settings and Preview pane sections, except that this Preview pane does not include sweep- and region-selection controls.

Figure 4‑57. Routine Review

Open this window from the Data Navigator by highlighting a Routine, and selecting the ‘View Routine’ command from a right-click menu or the ‘Available actions’ button, or by selecting the Routine Data Name in the Data Navigator Preview pane.

Routine Name Displays the Routine name

Activate Opens the Scope window loaded with these settings

Copy to Routine Pool Adds this routine to the loaded Routine Pool.

Routine Description Displays the Routine description

Display Signal Select the output signal to display

4.2.15 Scope (Reanalysis)

Figure 4‑58. Scope Reanalysis Window

This analysis version of the Scope window is used to display and reanalyze stored data. Both physical and virtual channels can be displayed here. Measurement regions are used as described for the Scope (acquisition) window. The additional window controls are described below:

Navigation pane:

The Navigation pane appears at the top of the Scope window. It displays an overview of the active signal’s full-scale data, with a gray box surrounding the magnification area.

Figure 4‑59. Navigation Pane

The Navigation pane “magnification” box can be used to scroll through the active signal’s data. Place the mouse cursor over the magnification box and it changes into a ‘hand’ icon; click and drag the magnification box to scroll through the data.

Run the defined analysis for the data series, and graph the results in the Analysis window. To stop a long-running analysis, click on the ‘Abort’ button in the bottom right corner of the main screen.

Open a special Reanalysis Measurements & Graphs dialog, where all edits apply instantly and interactively to the measurements and graphs, even during analysis. These dialog changes are temporary and override the loaded routine for quick interactive analysis.

Display any extra information (metadata) associated with the displayed data Series, such as the operator, preparation details, solution information, etc.

Open a Data Navigator window with all of your Experiment data and metadata available in a tree structure.

Layout

Create a new Layout window containing all Scope signals and Analysis graphs.

Center

Center the mean of the data in the selected signal pane. The Y-axis offset is automatically adjusted, while the Y-axis scaling is unchanged.

Autoscale

Autoscale all visible signals. Rescale the X-axes to their full sweep duration, and the Y-axes to their displayed sweeps data limits.

Persistence Display

Display all sweeps (per Preferences settings). Otherwise when disabled, only one sweep is displayed at a time.

Signal Display

Graphically arrange the input signals.

Stack: A vertical column of signals

Single: Only the active signal

m x n: A tiled array of signals

Sweeps Display

This button has 3 modes:

Sweeps

Each trace starts from time zero to the duration of the waveform.

Time Course

Display sweeps in time sequence on a single time axis. Portions without data are left blank (such as the time between triggered sweeps.)

Note: Emulation mode has a minimum 0.5 s interval between sweeps, both triggered and continuous. If the sweep duration is less than 0.5 s, the time between sweeps will be padded with “blank” time

Concatenated

Display sweeps similarly to the Time Course mode, but any blank portions are replaced by a vertical line

The ‘Show 3D view of current signal’ button brings up a separate 3D display window attached to the right of the Analysis window. The Sweep data are color-coded for amplitude, and their 3D graph can be rotated in any direction.

Sweep #:

The ‘Sweep #’ display at the bottom of the Scope window indicates the ‘active sweep’ number, the total number of sweeps in the Series, and either “all” or the total number of visible sweeps (per Preferences).

Table 4‑6: Scope Window Buttons

X- and Y-Axes Same as the acquisition Scope

Main window Additional items:

• 3D View

• Zero Baselines

• Show All Sweeps (with triggered sweeps)

• Select Sweeps (with triggered sweeps)

Data Additional items:

• Hide Sweep_# (with triggered sweeps)

• Show Sweep_# Only (with triggered sweeps)

4.2.16 3D View Window

The 3D View window creates a 3D representation of your data, color-coded to show amplitude variations.

The axis definition in 3D View is based on the change of a waveform over the course of successive sweeps. In a two-dimensional display, the X-axis represents the Sweep Time, while the Amplitude is plotted on the vertical Y-axis. For consistency, the vertical axis in the SutterPatch 3D view is also defined as the Y-axis. In the default orientation of the 3D View, the Z axis, on which the Sweep Number is plotted, points backward and to the right.

Figure 4‑60. 3D View

1. A “heat map” bar illustrates the color measurement units.

2. Magnification buttons are located in the upper right corner of the window.

A selection button allows X, Y, Z or “All” views to be selected for magnification.

Zoom In (magnify), Zoom Out (unmagnify), and Autoscale buttons are located here.

3. X, Y and Z axes limits can be set in the bottom section of the 3D View window. Their delta value is preserved when using the scroll bars to update the visual graph (and the numeric axes limits.)

4. The 3D graph viewing angle can be changed with a set of 3D buttons:

= Default View (X / Y / Z axes display)

X = Right View (Y / Z axes display)

Y = Top View (Z / X axes display)

Z = Front View (X / Y axes display)

Figure 4‑61. 3D Axis Definition

Alternatively, you can rotate the display in any direction by simply clicking and dragging the 3D graph. If you release the mouse button while dragging, the 3D display will rotate in the direction of the mouse drag.

4.2.17 Set Metadata

Preparation and stimulus parameters can be associated with an Experiment, Paradigm, or Routine as user-configurable “metadata”. Predefine the Metadata parameter values here. The number of available Metadata parameters can be configured in SutterPatch > Set Preferences > Metadata.

Show Summary An overview of the user-defined metadata parameters.

All parameters that have values defined are displayed, even if they would not be displayed based on the current setting of Set Preferences > ‘Metadata detail level’. Double-clicking on any line opens the Set Metadata dialog with the respective parameter selected.

Metadata Summary dialog

Metadata Parameter Parameter name

Current Value Parameter value

Increment Enabled If enabled, double-click to review details.

Prompt before Display metadata prompts before acquisition.

§ Expt Experiment

§ Pdgm Paradigm

§ Routn Routine

Metadata Group

Operator (Full detail level)

Preparation – Animal (Basic detail level)

Preparation – Tissue (Basic detail level)

Preparation – Cell (Basic detail level)

Experiment (Basic detail level)

Electrode (Extended detail level)

Recording Solutions (Extended detail level)

Paradigm (Full detail level)

Cell Health / Quality Control (Full detail level)

Series (+ Routine Data) (Full detail level)

Stimulus (Basic detail level)

Metadata Parameter Parameters from the selected metadata group are displayed for configuration (per the Set Preferences > Metadata detail level).

Each extended detail level Metadata Group includes a set of five user-defined parameters. Each user-defined Metadata parameter consists of a pair of fields, one for the name of the parameter, and one for its current value.

[ BASIC LEVEL ] The list of default metadata groups and their parameters:

Preparation – Animal

Animal Identifier An identifier for this specific animal

Animal Species Binomial species name

Animal Age The numerical age

Animal Age Units The units in which the age is given (e.g., h, d, m)

Animal Gender Eg., 1: f, 2: m, 3: undetermined

Animal Weight

Animal Weight Units The units in which the weight is given

Preparation – Tissue

Tissue Preparation Identifier An identifier for this specific tissue sample

Organ The organ which the sample came from

Preparation – Cell

Cell Preparation Identifier An identifier for this specific cell preparation

Cell Type The cell type

Cell Identifier An identifier for this specific cell

Cell Preparation Date ISO Date, Format: YYY-MM-DD

Cell Preparation Time Time of Day, Format: hh:mm[:ss.000]

Cell Preparation Incubation Duration

Cell Prep. Incubation Duration Units

Cell Preparation Incubation Temperature

Cell Prep. Incubation Temperature Units

Cell Preparation Incubation Solution

Ion Channel

Experiment

Experiment User Parameter 1 Name

Experiment User Parameter 1

Experiment User Parameter 2 Name

Experiment User Parameter 2

Experiment User Parameter 3 Name

Experiment User Parameter 3

Experiment User Parameter 4 Name

Experiment User Parameter 4

Experiment User Parameter 5 Name

Experiment User Parameter 5

Stimulus

Compound Identifier A code or identifier for the compound

Compound Name The name of the compound

Compound Concentration The concentration of the compound

Compound Concentration Units The units or the compound concentration

Light Stimulus Wavelength The wavelength of a light stimulus

Light Stimulus Intensity The intensity of a light stimulus

Light Stimulus Intensity Units The intensity units of a light stimulus

Mechanical Stimulus Intensity The intensity of a mechanical stimulus

Mechanical Stimulus Intensity Units The intensity units of a mechanical stimulus

Acoustic Stimulus Frequency The frequency of an acoustic stimulus

Acoustic Stimulus Intensity The intensity of an acoustic stimulus

Acoustic Stimulus Intensity Units The intensity units of an acoustic stimulus

Thermal Stimulus Temperature The temperature of a thermal stimulus

Thermal Stimulus Temperature Units °C, °F or K

Electrical Stimulus Frequency The frequency of an external electrical stimulus

Electrical Stimulus Intensity The intensity of an external electrical stimulus

Electrical Stimulus Intensity Units The intensity units of an external electrical stimulus

[ EXTENDED LEVEL ] Provides additional metadata groups and parameters:

Preparation – Animal

Animal Strain Strain, breed or variety characterizing the animal

Animal Genotype

Animal Preparation Date ISO Date, Format: YYYY-MM-DD

Animal Preparation Time Time of Day, Format: hh:mm[:ss.000]

Animal User Parameter 1 Name

Animal User Parameter 1

Animal User Parameter 2 Name

Animal User Parameter 2

Animal User Parameter 3 Name

Animal User Parameter 3

Animal User Parameter 4 Name

Animal User Parameter 4

Animal User Parameter 5 Name

Animal User Parameter 5

Preparation – Tissue

Organ Region A region or sub-structure within the organ

Preparation Method Preparation method for this sample

Tissue Preparation Date ISO Date, Format: YYY-MM-DD

Tissue Preparation Time Time of Day, Format: hh:mm[:ss.000]

Tissue Incubation Duration

Tissue Incubation Duration Units

Tissue Incubation Temperature

Tissue Incubation Temperature Units

Tissue Incubation Solution

Tissue User Parameter 1 Name

Tissue User Parameter 1

Tissue User Parameter 2 Name

Tissue User Parameter 2

Tissue User Parameter 3 Name

Tissue User Parameter 3

Tissue User Parameter 4 Name

Tissue User Parameter 4

Tissue User Parameter 5 Name

Tissue User Parameter 5

Preparation – Cell

Acutely Dissociated Cells

Cell Line

Slice Preparation

Whole-organ Preparation

In-situ Recording

Stem Cell Preparation

User-defined Preparation

Cell Dissociation Solution

Cell Preparation Dissociation Temperature

Cell Prep. Dissociation Temperature Units

Cell Fluorescent Marker

Cell Diameter

Cell User Parameter 1 Name

Cell User Parameter 1

Cell User Parameter 2 Name

Cell User Parameter 2

Cell User Parameter 3 Name

Cell User Parameter 3

Cell User Parameter 4 Name

Cell User Parameter 4

Cell User Parameter 5 Name

Cell User Parameter 5

Electrode

Electrode Identifier

Electrode Glass Manufacturer

Electrode Glass Item Number

Electrode Glass Ramp Test Value

Pipette Puller Manufacturer

Pipette Puller Model

Electrode User Parameter 1 Name

Electrode User Parameter 1

Electrode User Parameter 2 Name

Electrode User Parameter 2

Electrode User Parameter 3 Name

Electrode User Parameter 3

Electrode User Parameter 4 Name

Electrode User Parameter 4

Electrode User Parameter 5 Name

Electrode User Parameter 5

Recording Solutions

Solution Pair Identifier

Bath Solution Identifier

Bath Solution Name

Pipette Solution Identifier

Pipette Solution Name

Pipette Solution pH

Pipette Solution Osmolarity

Stimulus

Key Stimulus

Stimulus Duration

Compound Group

Compound Group Index

Compound Batch

Compound Lot

Compound Salt Code

Compound Solution

Compound Vehicle / Solubility Enhancer

Compound Vehicle Concentration

Compound Vehicle Concentration Units

Compound Reservoir Identifier

Application Tip Identifier

Compound Plate Identifier

Compound Plate Row

Compound Plate Column

Chem. Stimulus User Parameter 1 Name

Chem. Stimulus User Parameter 1

Chem. Stimulus User Parameter 2 Name

Chem. Stimulus User Parameter 2

Chem. Stimulus User Parameter 3 Name

Chem. Stimulus User Parameter 3

Chem. Stimulus User Parameter 4 Name

Chem. Stimulus User Parameter 4

Chem. Stimulus User Parameter 5 Name

Chem. Stimulus User Parameter 5

Light Stimulus User Parameter 1 Name

Light Stimulus User Parameter 1

Light Stimulus User Parameter 2 Name

Light Stimulus User Parameter 2

Light Stimulus User Parameter 3 Name

Light Stimulus User Parameter 3

Light Stimulus User Parameter 4 Name

Light Stimulus User Parameter 4

Light Stimulus User Parameter 5 Name

Light Stimulus User Parameter 5

Mechanical Stimulus User Parameter 1 Name

Mechanical Stimulus User Parameter 1

Mechanical Stimulus User Parameter 2 Name

Mechanical Stimulus User Parameter 2

Mechanical Stimulus User Parameter 3 Name

Mechanical Stimulus User Parameter 3

Mechanical Stimulus User Parameter 4 Name

Mechanical Stimulus User Parameter 4

Mechanical Stimulus User Parameter 5 Name

Mechanical Stimulus User Parameter 5

Acoust. Stimulus User Parameter 1 Name

Acoust. Stimulus User Parameter 1

Acoust. Stimulus User Parameter 2 Name

Acoust. Stimulus User Parameter 2

Acoust. Stimulus User Parameter 3 Name

Acoust. Stimulus User Parameter 3

Acoust. Stimulus User Parameter 4 Name

Acoust. Stimulus User Parameter 4

Acoust. Stimulus User Parameter 5 Name

Acoust. Stimulus User Parameter 5

Thermal Stimulus User Parameter 1 Name

Thermal Stimulus User Parameter 1

Thermal Stimulus User Parameter 2 Name

Thermal Stimulus User Parameter 2

Thermal Stimulus User Parameter 3 Name

Thermal Stimulus User Parameter 3

Thermal Stimulus User Parameter 4 Name

Thermal Stimulus User Parameter 4

Thermal Stimulus User Parameter 5 Name

Thermal Stimulus User Parameter 5

Electrical Stimulus User Parameter 1 Name

Electrical Stimulus User Parameter 1

Electrical Stimulus User Parameter 2 Name

Electrical Stimulus User Parameter 2

Electrical Stimulus User Parameter 3 Name

Electrical Stimulus User Parameter 3

Electrical Stimulus User Parameter 4 Name

Electrical Stimulus User Parameter 4

Electrical Stimulus User Parameter 5 Name

Electrical Stimulus User Parameter 5

Other Stimulus User Parameter 1 Name

Other Stimulus User Parameter 1

Other Stimulus User Parameter 2 Name

Other Stimulus User Parameter 2

Other Stimulus User Parameter 3 Name

Other Stimulus User Parameter 3

Other Stimulus User Parameter 4 Name

Other Stimulus User Parameter 4

Other Stimulus User Parameter 5 Name

Other Stimulus User Parameter 5

[ FULL LEVEL ] Even more metadata groups and parameters are provided

Operator

Full Operator Name

Preparation – Cell

Slice Preparation

Experiment

Experiment Category Parameter 1 Name

Experiment Category Parameter 1

Experiment Category Parameter 2 Name

Experiment Category Parameter 2

Experiment Category Parameter 3 Name

Experiment Category Parameter 3

Experiment Category Parameter 4 Name

Experiment Category Parameter 4

Experiment Category Parameter 5 Name

Experiment Category Parameter 5

Electrode

Electrode Glass Lot Number

Electrode Glass Material

Electrode Glass Item Outer Diameter

Electrode Glass Item Inner Diameter

Filamented Glass

Electrode Fire-polished

Electrode Coated

Electrode Coating Material

Electrode Beveled

Electrode Bevel Angle

Recording Solutions

Bath Solution Batch

Bath Solution Preparation Date

Bath Solution Preparation Time

Bath Solution pH

Bath Solution pH Adjustment Agent

Bath Solution Osmolarity

Bath Solution Osmolarity Adj. Agent

Pipette Solution Batch

Pipette Solution Preparation Date

Pipette Solution Preparation Time

Pipette Solution pH Adjustment Agent

Pipette Solution Osmolarity Adj. Agent

Liquid Junction Potential, computed

Liquid Junction Potential, measured

Paradigm

Bath Temperature

Bath Temperature Units

Ambient Temperature

Ambient Temperature Units

Atmospheric Composition

Atmospheric Pressure

Atmospheric Pressure Units

Atmospheric Humidity

Paradigm User Comment

Paradigm User Parameter 1 Name

Paradigm User Parameter 1

Paradigm User Parameter 2 Name

Paradigm User Parameter 2

Paradigm User Parameter 3 Name

Paradigm User Parameter 3

Paradigm User Parameter 4 Name

Paradigm User Parameter 4

Paradigm User Parameter 5 Name

Paradigm User Parameter 5

Cell Health / Quality Control

Cell Health User Parameter 1 Name

Cell Health User Parameter 1

Cell Health User Parameter 2 Name

Cell Health User Parameter 2

Cell Health User Parameter 3 Name

Cell Health User Parameter 3

Cell Health User Parameter 4 Name

Cell Health User Parameter 4

Cell Health User Parameter 5 Name

Cell Health User Parameter 5

Series (= Routine Data)

Routine User Comment

[ Parameter description ]

Configuration choices for the selected parameter:

· Do not write this parameter This parameter is not stored with recorded data.

If this parameter was previously written in this Experiment, then its Previous Value is displayed.

· Use last value The last used parameter is stored with recorded data.

· Use a previous value Select from a drop-down list of previous parameters.

· Use new value Enter a new value for the metadata parameter.

· Increment Numerically increment the parameter

· Experiment At the start of each Experiment

· Paradigm At the start of each Paradigm

· Routine At the start of each Routine

Prefix Enter text to be prepended to the value

Start value The initial value (including decimals and negative numbers)

Increment: Select an arithmetic operator [ +, -, *, / ]

[ ] Enter the incremental amount

Suffix Enter text to be appended to the value

Prompt for confirmation (before:)

· Experiment At the start of an Experiment

· Paradigm For “planned” or named Paradigms (i.e., not auto-triggered by Routines)

· Routine At the start of every Routine

Confirm Metadata Settings This dialog displays when an Experiment, Paradigm or Routine is started while enabled for prompts.

Write The metadata parameter is stored with the Experiment, Paradigm or Routine

Metadata Parameter The parameter to update

Next Value The value to write

Update Apply to future Experiments, Paradigms or Routines

Prompt Enable/disable individual prompts

4.3 General

SutterPatch general operations.

4.3.1 Command Window

The Command window is labeled with the currently loaded Experiment filename. A history of commands and responses is displayed in the upper portion of the window. Some warning messages are also displayed here

This window is also an Igor Pro code interpreter, for programmatic interaction with SutterPatch. Commands can be entered into the command buffer in the lower portion of the window. A maximum of 400 characters can be entered into the command buffer, however they can be spread over multiple lines. Highlighted lines in the history section are transferred into the command section for processing when the Enter key is pressed.

The Command window has a resizing line between the upper history section and the lower command section – the mouse cursor will change to a double-headed arrow.

For more information, see the Programming chapter of the Igor Pro manual.

4.3.2 Dashboard Panel

The Dashboard panel provides a convenient gateway to key areas of the SutterPatch program:

Figure 4‑62. SutterPatch Dashboard

· Acquire Data Live recordings and acquisition configuration

· Analyze Data Review and analyze data in the Data Navigator

· View Last Data Open the Experiment’s last recorded data Series

· Set Metadata Configure user-defined metadata parameters

· New Experiment Start a new Experiment, and/or switch the amplifier model or emulation mode

Clicking the Acquire Data icon opens an adjoining secondary pane:

Figure 4‑63. Dashboard – Acquire Data

· Control Panel Hardware control via the Amplifier Control Panel

· Membrane Test Monitor seal formation and cell health

· Free Run Run an oscilloscope-style signal monitor

· Routines Configure Routine acquisition settings

· Paradigms Control the execution of commands

· Set Metadata Set user-defined metadata parameter values

4.3.3 File Import/Export

Experiments & Data

A SutterPatch Experiment is saved as a file (*.pxp) that includes raw data, analyses, graphs, routines, paradigms, etc. Saved Experiments can then be re-opened, or merged with the current Experiment via the Data Navigator ‘Import’ button.

If a SutterPatch Experiment file is opened into Igor Pro 7 without SutterPatch running, its graphs will not be visible - you will need to use the Igor Pro 7 Windows / Graphs’ or ‘Windows / Layouts’ menu items to display them.

Series-level (Routine) data can be exported to the pCLAMP ATF file format via the Data Navigator ‘Available Actions’ button (or right-click menu). One Series produces one ATF file.

Graphs and Layouts

Import or export graphs for the current experiment via the Analysis Editor / Files options. Graph files are stored as Igor Binary Wave (*.ibw) files.

Note: Graph data for each axis can also be saved as Igor Pro 6 one-dimensional wave files, however files using this older format cannot be re-imported back into SutterPatch.

Graphs can be exported to a Layout window via:

Analysis Editor: [ To Layout ] button

Scope window : "Export a layout" button

Paradigms: Export step

Graphs exported to the Layout window use the Preferences / Export Graphics settings.

Individual graphs can be saved as Graph Macros - recall them via the Windows / Graph Macros menu.

Layout windows can be saved as Layout Macros - recall them via the menu Windows / Layout Macros menu.

Routines.

PatchMaster Pulse Generator Files (*.pgf) can be opened in the Routine Editor ‘Pools and Files’ section and their Sequences merged with the current routine pool.

Templates

Templates can be imported or exported via the Template Editor as Igor Binary Wave (*.ibw) files.

4.3.4 Log Window

The Log window collects time-stamped commands, responses, administrative information and error messages in a structured, searchable format. These entries provide a history of the steps having a possible influence on the execution of the experiment and its data.

When SutterPatch starts up, the Log window displays the SutterPatch Version and Build numbers.

Column Names (default) The Log window can be sorted based on a column – click on a column header name, and the Log Window is sorted numerically and alphabetically based on that column. Clicking on the column header a second time reverses the sort order.

Date / Time The Date/Time-stamp is in ISO format:

Year-Month-Day hours:minutes:seconds.milliseconds

Example: 2016-04-25 14:21:34.478

Note: The timestamp has a resolution of 1 ms, and multiple entries can occur within this timespan. This may create an unreliable sort order when sorting by Date/Time.

To sort Log entries in the order they were created, right-click on the column header and select Default View. Alternatively, right-click on a column header, choose ‘Select columns to show’, enable the Log ID column, and then sort on this column.

Event Type Log entries are assigned an Event Type:

Command Command window execution

Comment

Data Acquisition Scope acquisition operations

Data Management

Exit

History

Metadata

Paradigm

Routine Routine Pool operations

Startup Startup / loading information

Unknown

Event Parameter A text description of the log entry.

Paradigm Data / Series Paradigm, Routine and Series (data) names associated with the log entry are displayed

Experiment Category The Experiment Category values set in the Metadata.

These are properties that help in organizing experiments, such as a hierarchical category structure:

Experiment Category 1: Site

Experiment Category 2: Department

Experiment Category 3: Lab

Experiment Category 4: Project

Experiment Category 5: Screen

Column Names (additional)

User Name The name of the user logged onto the computer.

Host Name The name of the computer.

OS & Version The operating system, its version number and information about build or Service Pack, if applicable.

Software & Version The SutterPatch application name, version and build number.

Log ID A unique sequence number for events. Useful for sorting events that occur within the same 1 ms Time stamp.

Column header (right-click for menu)

Search for Text: A Search Log dialog opens with multiple full-text search options.

Find Enter or select the text string to search on.

Case sensitive Text capitalization must match.

Whole word Partial-word matches are ignored.

Select columns to search

All Search all columns in the Log window.

Previous Revert to the initial column selection

Column list Select columns by clicking on them. The <Shift> and <Ctrl> or <Cmd> keys for multi-row selection are supported, according to operating system standards.

· Highlighted Display all rows, with matches highlighted.

· Filtered Only display rows that match the Find criteria.

Back Select the previous matching row.

Next Select the next matching row.

Wrap Cycle between the first and last search matches when the Back/Next buttons are used.

Clear Return the Search window to its default settings.

Find Execute the search.

Select columns to show: Enable or disable the display of columns.

Sort on this column: Alphabetically sort the entire window based on the selected column.

Default view: Restore the Log window default view. All data sorting, filtering, and column selections are cleared.

Paradigm / Routine / Data (Right-click menu items)

· Paradigm data

Review Paradigm Data: Opens the Paradigm data into a Paradigm Review window.

· Series data

Analyze Series: Opens the named Series data into a Scope window for analysis.

4.3.5 Menus

The SutterPatch main menu item contains all of the SutterPatch-specific menu items. The rest of the main menu items provide the standard Igor Pro functionality. For documentation of the non-SutterPatch features, refer to the Igor Pro online help or manual.

Window/Dialog Controls

Keyboard “Return” key = ‘OK/Yes’ buttons

Keyboard ESC key = ‘Cancel’ button

File

New Experiment Unload the current Experiment and start a new Experiment.

It is recommended that you create one Experiment per cell, to keep file sizes manageable.

Note: Even if immediately selected after saving an Experiment, you will still be re-asked to save the prior Experiment (due to internal processes.)

Open Experiment Open a previously saved SutterPatch Experiment (*.pxp) file. If a SutterPatch experiment is opened into an Igor-only session, SutterPatch is automatically loaded.

Save Experiment If the current experiment is already named, it is immediately saved. Otherwise, a ‘Save experiment as’ file dialog is displayed. If Preferences are enabled for automatic file naming, a default Experiment name is provided.

Save Experiment As Saves and renames the loaded Experiment to a new file name.

Save Experiment Copy Save the Experiment to another file without closing the current session

Revert Experiment Undo any unsaved changes to the Experiment

Recent Experiments A list of recently used Experiments

Exit An Experiment file ‘Save’ dialog is displayed before closing the program. If an Experiment is not saved, global variables and window sizes/positions are lost.

Data

Data Browser Access all SutterPatch objects contained in the experiment.

Windows

The Windows menu provides access to graphs, tables and layouts.

Layout

The Layout menu only displays when a Layout is the active window. Use it to modify the Layout window objects and display.

SutterPatch

Dashboard Display icons for core program functions

Scope Window Bring an open Scope window to the front.

Hardware Control

Amplifier Control Panel Open the hardware control panel

Set Seal Test Set the amplitudes of the Seal Test pulses

Reset Control Panel Return the Amplifier Control Panel to its default settings

Reset USB Re-initialize USB communication with the computer. If in Demo mode, you need to start a ‘New Experiment’ to access ‘Reset USB’.

Membrane Test Open and run the Scope window to monitor seal formation and cell health

Free Run (Scope) Open and run the Scope window in oscilloscope style

Reset Acquisition Stop the Paradigm and/or data acquisition and clear corrupted acquisition settings

Paradigm Editor Open the dialog to load, edit and run Paradigms

Routine Editor Open the dialog to load and edit Routines

Template Editor Open the dialog to manage templates

Equation Editor Open the dialog to load and edit Equations

Solution Editor Open the dialog to control solutions

Camera Module Open the window to capture images

Data Navigator Open the window to organize and display the experiment Paradigm, Routine and acquisition data in a tree structure

Analysis Editor Open the dialog to manage analysis graphs

Set Metadata Open the dialog to configure user-specified experimental information

Set Preferences Open the dialog to modify the default program settings

Log Window Open the window to display a history of program actions

Shortcuts Open the Shortcuts Editor dialog to manage keyboard shortcuts

Scope Right-click Menus

Different areas of the Scope windows support additional functionality through "right-click" menus in Windows, or "Command-click" menus in macOS.

Scope (Acquisition) main window

· Autoscale All Axes

· Add Annotation

· Export Graphics Copy the selected signal to a Graph window

· Colors

· Hide Signal ‘Signal Name’

· Show Signal ‘Signal Name’ Only

· Stack All Signals

Scope data (right-click on the data)

· Browse ‘Series_Signal name’

· Edit ‘Series Signal name’

· Remove Sweep_#

· Duplicate Trace

· Replace Wave

· Copy

· Modify Sweep_#

· Customize

· Mode

· Line Style

· Line Size

· Markers

· Marker Size

· Color

· Bring to Front

· Send to Back

· Forward

· Backward

· Modify Contour

· Modify Image

· Quick Fit

· Export Graphics

· Hide Signal ‘Signal Name’

· Show Signal ‘Signal Name’ Only

· Stack All Signals

· 3D View

· Zero Baselines The average of the first four data points is sub-

tracted from the sweeps display. Does not affect data values in measurements.

· Hide Sweep_#

· Show Sweep_# Only

· Show All Sweeps

· Select Sweeps

Scope Y-Axis

· Autoscale All Axes

· Autoscale Axis

· Full Scale Axis

· Axis Properties

· Hide Signal ‘Signal Name’

· Show Signal ‘Signal Name’ Only

· Stack All Signals

Scope X-Axis

· Autoscale All Axes

· Full Scale Axis

· Axis Properties

Scope (Reanalysis) main window Additional items vs. Scope (Acquisition)

· 3D View

· Zero Baselines The average of the first four data points is sub-

tracted from the sweeps display. Does not affect data values in measurements.

· Show All Sweeps

· Select Sweeps

4.3.6 Preferences

Preferences settings customize the default settings for several areas of the SutterPatch program:

§ Acquisition

§ Display

§ Files ~~path~~

§ Graphics ~~export~~

§ Hardware

To access, go to the SutterPatch/ Set Preferences menu.

Figure 4‑64: Preferences Settings

i. General

Enable drag and drop for routine and paradigm editors

The Routine Editor loads a “pool” of Routines from a Routine Pool file. These Routines can be re-arranged in the Routine Pool list by clicking and dragging with the mouse. The Paradigm Editor operates in the same manner, and also displays a list of Paradigm Steps, which can also be re-arranged by dragging-and-dropping.

Copy “To Clipboard” graph format

Several popular file formats are supported:

· PNG Portable Network Graphics

· PDF Portable Document Format

· TIFF Tagged Image File Format

· JPEG Joint Photographic Experts Group

Note: When pasting, not all formats may be supported by other programs.

Automatic window repositioning.

When SutterPatch windows or dialogs are opened or moved, they are repositioned so that they are fully visible. If a “child” window is opened, the parent window is moved to the left until the child sub-window is fully visible or the parent window reaches the left edge of the main window/screen.

Note: You can also bring all unminimized windows into view with the Windows > Control > Retrieve All Windows menu.

Dual-monitor option (macOS only)

One screen Prevents windows spanning across monitors.

If a “parent” window is moved to another monitor, it fully displays in the new monitor, while any child sub-window remains behind fully displayed in the original monitor.

Preferences Defaults Restore default preferences settings for all preferences.

ii. Files and Naming

Data files path: Browse to select a folder

Enable automatic naming for experiment Name new experiments as below…

Experiment file name example: (Maximum 34 characters)

Text: Legal characters are A-Z, a-z, 0-9 and “_”. Include user text in the file name

Date: YYMMDD Include the date in the file name

Time: hhmmss Include the time in the file name

ii.

iii. Scope

To apply these settings, open or re-open the Scope window.

General

Time axis unit:

· Auto-set Sweep duration < 120 s, use “s”

Sweep duration >= 120 s, use “min”

Sweep duration >= 7200 s, use “h”

· SI unit Always use “s”

Acquisition

Y axis autoscale settings:

· Autoscale The Y-axis limits are rescaled with each sweep so that all data are visible

· Autoscale from zero One Y-axis limit is positioned at zero, and the other Y-axis limit is rescaled with each sweep. The Y-axis direction matches the sign of the largest absolute value in the sweep, can change during a sweep, and can be different for each sweep.

If Persistence is enabled, the direction of the first sweep is used for all subsequent sweeps.

Autoscaling is just a display setting, - all data, both positive and negative, are recorded.

· Full scale The full-scale range is used

Signals Display

· Show all signals

· Use last selection

· Signals [ ] Enter signal numbers and/or signal ranges separated by commas

For example: 1,2,4-5

Reanalysis

Show tags by default

Y axis autoscale settings:

· Autoscale The Y-axis limits are rescaled with each sweep so that all data are visible

· Autoscale from zero One Y-axis limit is positioned at zero, and the other Y-axis limit is rescaled with each sweep. The Y-axis direction matches the sign of the largest absolute value in the sweep, and can change during a sweep, and can be different for each sweep.

If Persistence is enabled, the direction of the first sweep is used for all subsequent sweeps

Autoscaling is only a display setting - all data, both positive and negative, are recorded

· Full scale The full-scale range is used

Signals Display

· Show all signals

· Use last selection

· Signals [ ] Enter signal numbers and/or ranges separated by commas

For example: 1,2,4-5

Sweeps Display

· Show all sweeps

· Use last selection

· Sweeps [ ] Enter sweep numbers and/or ranges separated by commas

For example: 1,3,5-20

Appearance Use a color palette to choose colors

Active signal panel color [ light gray ]

Inactive signal panel color [ dark gray ]

Active sweep color [ blue ]

Inactive sweep color [ red ]

Drop-down color palette Click on a color square to set it as the active color

Other Use a color dialog with more options

Opaque < unused >

[ Preview Pane ] The selected signal and sweep colors are displayed in a preview pane

Tip: For dark-room experiments, the window background color can be adjusted by the operating system:

· Windows: In the Control Panel / Appearance / Personalization window, scroll down and select the High Contrast Black theme, or use the Windows Magnifier tool with option ‘Turn on color inversion’ enabled.

· macOS: Press ‘Control-Option-Command-8’ to set the System Preferences / Accessibility / Display / Invert Display colors option, or open its menu with ‘Command-Option-5’.

ii.

iii.

iv. Membrane TestMembrane Test

To apply these settings, open (or re-open) the Membrane Test Scope window. These settings apply to all connected IPA headstages.

Sampling Rate:

· 20 kHz (50 µs)

· 50 kHz (20 µs)

A 20 kHz sampling rate is more than sufficient for most situations. However, if the Amplifier Control Panel input filter is increased to 20 kHz, then also increase the Membrane Test speed to its 50 kHz (20 µs) sampling rate to prevent aliasing.

Duration:

· LONG (200 ms) 200+ ms start-to-start timing

· SHORT (20 ms) 100+ ms start-to-start timing

Two Membrane Test sweep durations are provided. The pulse duration is 50% of the sweep duration.

Line Frequency Reduction:

· Off

· 50 Hz

· 60 Hz

Alternating Current (AC) power contains 50 or 60 Hz oscillations that can cause sinusoidal line frequency noise or “hum” to appear in recorded signals. This option reduces such noise by ~90% over 6 harmonics. The adjusted data is displayed in the input signal in real time.

ii.

iii.

iv.

v. Export Graphics

These settings apply to Layout windows and stand-alone graph windows (not graph files).

Layouts

Default graphs per page

· 1

· 2

· 3

· 2 x 2 (column x row)

· 2 x3 (column x row)

· 2 x 4 (column x row)

Graphs

left (typically the Y-axis)

Tick location:

· Outside

· Crossing

· Inside

· None

bottom (typically the X-axis)

Tick location:

· Outside

· Crossing

· Inside

· None

Labels:

· On

· Axis only

· Off

Grid:

· Off

· On

· Major only

ii.

iii.

iv.

vi. Hardware

Prompt on startup (if no Sutter hardware is found).

When a new Experiment is started, if Sutter patch-clamp hardware is not connected to the computer and turned on, you are prompted to retry the USB connection or select an emulation mode.

If no Sutter hardware is found, emulate:

· IPA Integrated Patch Amplifier system

· Double IPA Dual-headstage IPA system

When a new Experiment is started, if Sutter patch-clamp hardware is not connected to the computer and turned on, automatically start up in the selected hardware emulation mode.

Routine Conversion for Amplifier Compatibility

When a routine is loaded, it is checked for compatibility with the attached hardware, and the selected action is taken:

· Prompt user When an incompatible routine is loaded, a dialog queries which conversion type to use

· Duplicate and Convert Create a copy of the routine, convert it for hardware compatibility, and increment its name

· Convert only Convert the Routine for hardware compatibility

· Do not Convert The routine is not converted

Output zero calibration

[ Calibrate Now ] User calibration trims the DAC to remove its microvolt bias from the output stimulus (typically ~50 µV)

Caution: Do not calibrate during a recording, as the holding potential is reset to zero.

[ Reset to Zero ] Remove all calibration.

Useful for current-clamp recordings from extremely high-resistance cells, when you cannot afford to have any current injected into the cell. This feature eliminates any currents due to trimming the DAC.

Or, if switching between multiple Sutter amplifiers, you might not want to use the calibration value of one amplifier for another.

ii.

iii.

iv.

vi.

vii. MetadataMetadata

Metadata detail level: Increasing levels of data categorization complexity.

Select which metadata groups and parameters are visible for configuration in Set Metadata and in Data Navigator / Build Hierarchy.

1 Basic Show only the most essential parameters

2 Extended Show additional detail

3 Full Expose all available metadata parameters

Note: This setting does not affect the Metadata Review dialog - all user-defined metadata are displayed irrespective of the metadata detail level.

ii.

iii.

iv.

vi.

vii.

viii. Factory Reset

Warning! When you do a factory reset, you will lose ALL your configuration data (including Metadata, Scope, Preferences, etc.)

[ Factory Reset ] Click this button to reset SutterPatch to it default settings. SutterPatch will need to be exited and restarted to complete the factory reset.

4.3.7 Shortcut Editor

Keyboard control of SutterPatch is available by configuring keyboard shortcuts.

Figure 4‑65: Shortcuts Editor

Control Key Assignments / Command Key Assignments

A Control Key shortcut assignment can be changed by clicking on its drop-down list and selecting a new menu item.

· Windows ‘Control’ key: CTRL

· macOS ‘Command’ key:

Use a shortcut Control Key by holding down the ‘CTRL’ or ‘Command’ key while simultaneously clicking its assigned number.

A list of the Control Keys and their default SutterPatch menu items:

Control-1 Show Dashboard

Control-2 Show Scope Window

Control-3 Amplifier Control Panel

Control-4 Membrane Test

Control-5 Free Run (Scope)

Control-6 Paradigm Editor

Control-7 Routine Editor

Control-8 Data Navigator

Control-9 Set Metadata

Control-0 Log Window

Other SutterPatch menu items available for shortcut assignment:

Reset Acquisition

Equation Editor

Template Editor

Analysis Editor

Shortcut Editor

Camera Module

Event Detection

Set Preferences

Function Key Assignments

Computer keyboards usually include a set of Function keys for special actions. Configure a ‘Function Key’ shortcut assignment by clicking on its drop-down list and selecting a new menu or action item.

Function Keys:

F1 Igor Pro Help function < reserved >

F2 – F12 not assigned < available >

On some keyboards, you also need to press the ‘Fn’ key to use function keys.

On some Windows systems, F12 is reserved for debugging.

macOS reserves nearly all function keys for itself. In order to use function keys for an application, you must first check a checkbox in the Keyboard control panel. Even then, macOS will intercept some function keys.

Reset Key Assignments

Reset all Control and Function Key Assignments to their default assignments.

Shortcut Actions

Additional custom keyboard Actions can be created and their shortcuts managed.

Note: Shortcut Actions key combinations that include a Function or ‘Ctrl / Cmd’ key are automatically added to the SutterPatch / Shortcuts submenu.

Shortcut Pool Files

New Shortcut Pool Create a default Shortcut Pool

Load Shortcut Pool Load the Shortcuts of a previously saved Shortcut Pool file into the Shortcut Pool

Revert to Last Saved Undo any unsaved changes to the Shortcut Pool

Save Shortcut Pool Save the Shortcut Pool using its existing file name and path

Save Shortcut Pool As… Save the Shortcut Pool to a new file, and switch to the new file. The default file name is the same as the original file name.

Save Shortcut Pool Copy… Save the Shortcut Pool to a new file, but do not switch to the new file. The default file name has ‘Copy of’’ prepended to it.

Merge Shortcut Pools Insert the Shortcuts from a previously saved Shortcut Pool file into the loaded Shortcut Pool

[ Shortcut Pool file path ]

Columns

On Enable/disable the Shortcut Action

Key The assigned keyboard key.

Modifier The keyboard “modifier key” used in a key combination

Windows

· CTRL Only use with keys ‘0 – 9’

· ALT Keys ‘0 – 2’ reserved by Igor for File / Recent Experiments.

· SHIFT

· FUNC

· CAPS LOCK Works for upper- and lower-case keys

macOS

· Command Only use with keys ‘0 – 9’

· Option

· Shift

· Control Keys ‘0 – 2’ reserved by Igor for File / Recent Experiments.

· Caps Lock Works for upper- and lower-case keys

Shortcut Action An Action’s instructions and settings

Click a field in the pool to highlight an Action and make it the active entry for the following buttons. Click-and-drag a field to reposition an Action in the pool.

Insert Add an Action to the ‘Shortcut Action’ list and open the Shortcut Actions Editor for setup.

These Actions operate the same as Paradigm steps, with additional ‘Paradigm’, ‘Start Acquisition’ and ‘Stop Acquisition’ Actions:

Amplifier

Analysis

Camera

Execute

Export

Front Window

Hide Window

Paradigm Stop, Pause, Resume, <list of loaded Paradigms to run>

Reset Timer

Routine

Scope Operation

Set Axis

Set Checkbox

Set Variable

Set Tag

Start Acquisition in an open Scope acquisition window

Stop Acquisition in an open Scope acquisition window

View Last

Write Log

Assign Key This button opens the Shortcut Key Input dialog (or double-click in a “Key” or “Modifier” field) to input the desired keyboard combination for a letter, number, or symbol.

Note: Available keyboard letters, numbers, and symbols can vary from computer to computer, depending on the computer OS and Igor Pro’s key usage. (Reserved keys typically open another window type, or are non-responsive.)

Tips: If the CAPS LOCK button is on when assigning a key, the key is case insensitive.

Keyboards often have a Function (FN) button to allow special access to the Function keys.

Although the F1 function key is reserved in Igor Pro, it can be assigned if used with a modifier key.

Delete Key Remove the Key entry for the selected Action.

Edit Open the Shortcut Editor dialog (or double-click in a “Shortcut Action” field) to change the Action’s parameters.

Execute Run the selected Action

Delete Remove the selected Action from the ‘Shortcut Action’ list

Save Saves any changes to the current Shortcut Pool file.

4.3.8 Sample Files

Sample files (subject to change) are included with the SutterPatch application:

1. SP_EquationPool.txt A sample equation pool

1. X3pi

2. ElapsedTime

3. Temperature

2. SP_ParadigmPool.spp A sample paradigm pool file

1. Amplifier_Setup Set initial amplifier settings

2. Start_one_Series Start acquisition of one routine

3. Set_amplifier_and_start_IV Set amplifier to a known state, then start a routine

4. Start_two_Series Start acquisition of two subsequent routines

5. Start_ForEachSweep Start acquisition of a routine, individually triggering each sweep

6. Interactive_acquisition_1 Run an interactive acquisition stopping at a given analysis condition.

7. Interactive_acquisition_2 Run an interactive acquisition using 2 checkboxes: Checkbox-4 selects between 2 routines, checkbox-2 stops.

8. Toggle_Persistence An example paradigm using Checkbox-6 to toggle scope persistence while acquiring a routine

9. Switch_amplifiers Switch between multiple amplifiers

10. Tuning_with_Keys Use the keys “,” and “.” to decrement and increment the Routine Variable-1 by 10 mV

11. Tuning_with_Input Use the paradigm "Input" control to increment and decrement the Routine Variable-1

3. SP_RoutinePool.spr A sample IPA routine pool file

1. IV I/V for voltage-clamp mode

2. IV_Continuous I/V with continuous acquisition

3. IV_tuning I/V for “tuning” paradigm

4. Synaptic_Stim Synaptic stimulation

5. Multi_Test Multi-channel waveform

6. Bowtie_Test Multi-channel waveform

7. IV_P4 I/V with 4 leak pulses

8. SS_Inactivation Steady-state inactivation

9. Recovery_Inactivation Recovery from inactivation

10. Onset_SlowActivation Onset Slow activation

11. SS_SlowInactivation Steady-state slow activation

12. Recovery_SlowInact Recovery from slow inactivation

13. Test_Pulse Test pulse

14. ContinuousNoOut Acquisition without any output waveform

15. Amplitude_Equations Equations for waveform ouput

16. TemplateTest Template wave for waveform output

17. Template_SponAct Template wave from a recorded signal

18. TemplateAndVirtual Template wave and recording virtual signals

19. IV_CC I/V for current-clamp mode

20. SynapticStim_PlusDig Synaptic stimulation with 50 ms depolarizing step, digital output, and Rs measurement

4. SP_RoutinePool_DIPA.spr A sample Double IPA routine pool file

1. IV I/V for voltage-clamp mode

2. IV_Continuous I/V with continuous acquisition

3. IV_tuning I/V example for “tuning” paradigm

4. Synaptic_Stim Synaptic stimulation

5. Multi_Test Multi-channel waveform

6. Bowtie_Test Multi-channel waveform

7. IV_P4 I/V with 4 leak pulses

8. SS_Inactivation Steady-state inactivation

9. Recovery_Inactivation Recovery from inactivation

10. Onset_SlowActivation Onset Slow activation

11. SS_SlowInactivation Steady-state slow activation

12. Recovery_SlowInact Recovery from slow inactivation

13. Test_Pulse Test pulse

14. ContinuousNoOut Acquisition without any output waveform

15. Amplitude_Equations Equations for waveform ouput

16. Template_SpontAct Template wave from a recorded signal

17. TemplateTest Template wave for waveform output

18. TemplateAndVirtual Template wave and recording virtual signals

19. IV_VC_CC IV for voltage- and current-clamp modes

20. A_T_InactRec_P4 Inactivation with leak subtraction

5. SP_TemplatePool.spt A sample template pool file

1. minis

2. R1_S1_10minSpontAct

3. Template1_Sweep5

4.3.9 Startup

The SutterPatch application startup sequence:

1. Click on the Igor Pro 7 icon to launch SutterPatch.

2. A “splash” screen displays file opening information, and a blank Experiment window is opened.

3. Next, a larger splash screen displays to choose between starting SutterPatch or opening Igor Pro without it.

4. After clicking ‘Start’, the splash screen displays a progress bar for file compilation.

5. Then a ‘Save experiment as” dialog displays a standard file saving dialog to name the Experiment file.

6. If a Sutter system is not attached or not communicating with the computer, the ‘No USB Connection’ pane allows you to try re-establishing the USB connection(s), or to select a hardware-emulation demonstration mode (IPA, Double IPA).

In “demo” mode, all functions are available, except that the input and output channels use simulated data, and the Amplifier Control Panel and acquisition Scope window are labeled with “Demo”.

7. The Dashboard panel is displayed. Additional SutterPatch windows might open, if they were displayed in the prior experiment.

5. Programming

5.1 Data Format

SutterPatch data are written in a 64-bit double-precision binary floating-point format. This supports a decimal precision of 17 significant digits.

The data are stored within an Igor Experiment (*.pxp) file.

For large data sets, an optional HDF5 file format will be available for streaming data acquisition without resaving the experiment at the end of a recording.

5.2 Data Structure

SutterPatch recorded data are stored as multidimensional data waves, and are listed per signal in the Data Browser. Select a data wave in the Data Browser and right-click to ‘Edit’ the Signal data in a spreadsheet-style table. The two-dimensional data wave is displayed with one row per sample point and one column per trace, with the number of data table columns increasing with the number of sweeps.

Warning! The raw data can be directly edited in the Data Browser – this is not recommended, as it permanently alters the data.

Note: While SutterPatch does not read the older Igor one-dimensional wave data-format, graph data for each axis can be separately exported to it. See the Analysis Editor / Files menu.

5.3 Data Paths

The Data Browser path uses a “root” of ‘SutterPatch’. The Data Browser right-click ‘Copy Full Path’ command copies a Signal’s data wave path to the clipboard.

Note: This path references Igor’s internal data folders, and not the computer OS file system.

The object’s path name can be used in user functions and executable commands. However, when referencing an active Scope window, the path name to the data wave can be substituted by “t[#]”, where ‘#’ refers to the signal position number in the Scope window.

5.4 User Functions

SutterPatch functionality can be extended through the use of user-defined Functions.

To create a user Function:

1. Open the menu for Windows / Procedure Windows / Procedure Window.

2. Enter your user code into the Procedure window, following its ‘#pragma’ and ‘#include’ lines.

Example:

#pragma TextEncoding = “Windows-1252”

#pragma rtGlobals=3 // Use modern global access method…

#include “SP_Globals”, optional

Function SayHello()

DoAlert 0, “Hello World!”

End

Note: The Function name must include trailing open/close parentheses “()”

3. Click on the Compile button at the bottom of the window.

4. Enter the Function name (including parentheses) into the Command window and press ‘Enter’, or use it in a Paradigm ‘Execute’ step.

For more information on creating your own functions, see the Igor Help topics on Programming / User-Defined Functions, and Procedure Windows.

Warning! User-defined functions only exist during the Experiment. They are not stored when the Experiment is closed. If you plan to re-use them in other Experiments, save them to a separate file, such as with a word processor.

Also, while user-defined functions are stored internally by Igor, there is no visible list, so you will need to maintain such a list manually.

6. MAINTENANCE

6.1 Inspection

Periodically inspect all cables and connections to make sure that all cables are sound and that all connections are firm and evenly seated.

Warning! Do not hot-swap headstages. Turn off the IPA power before plugging/unplugging headstages.

6.2 Cleaning

Routine cleaning of the IPA system is required to prevent excessive dust accumulations. Wipe all exterior surfaces with a dry, soft, cotton cloth.

6.3 Calibration

The IPA amplifier can be calibrated in the Set Preferences / Hardware section:

Calibrate Now Trim the unit’s DAC (Digital-to-Analog Converter) to remove its microvolt bias from the output stimuli.

Caution: Do not calibrate during a recording, as the holding potential is reset to zero.

Reset to Zero In case of problems with the DAC trimming, removes all calibration.

7. TROUBLESHOOTING

7.1 Technical Support

Technical support is provided to customers at no charge.

Support hours: 8:00 AM - 5:00 PM PST (Pacific Standard Time).

Telephone: (+1) 415.883.0128

Fax: (+1) 415.883.0572

E-mail: info@sutter.com

Address: Sutter Instrument Company

One Digital Drive

Novato, CA 94949

When contacting us for technical support, please provide your SutterPatch version and “build” numbers to help us troubleshoot your situation. These numbers are found in the Start splash screen during program loading, or in the Log Window Startup events.

For issues regarding Igor Pro features (all non-SutterPatch menu items), please contact Wavemetrics, Inc. for technical support.

7.2 Startup Issues

7.2.1 Installation Fails

Problem: The SutterPatch installation on Windows fails due to language pack incompatibilities.

Solution: Support for foreign language packs has been added. Should language versions still cause problems, please contact Sutter Technical Support.

7.2.2 Application Not Loading

Problem: The SutterPatch application does not load – the startup sequence only loads Igor Pro.

Solution: If available, execute the Igor Pro menu command ‘Macros / Autocompile’.

7.2.3 Startup Errors

Problem: When starting up SutterPatch after installing a software update, program errors display.

Solution: Closing and re-opening SutterPatch sometimes clears up these errors.

7.3 Acquisition Issues

7.3.1 Acquisition Locked

Problem: The Scope window, Routine Editor or Paradigm Editor lock up during acquisition.

Solution: Use the menu command SutterPatch / Reset Acquisition to unlock acquisition.

Some older computers might be too slow – reduce the 10 kHz output sampling rate in the Routine ‘Acquisition & Routine Parameters’.

Disable the screen saver and any Power Save or Sleep modes. Refer to the documentation of your operating system for details.

Software for certain license protection keys (dongle) sends commands to all USB ports on a computer, which may interfere with operation of the IPA system.

7.3.2 Channel Saturated

Problem: A Scope window channel is completely saturated during acquisition.

Solution: The corresponding headstage is not attached to its port. Power off the IPA system and reconnect the headstage cable.

Note: The headstage HDMI connectors do not lock on - make sure they do not disconnect from their port.

7.3.3 Headstage Noise I

Problem: The noise levels of the instrument suddenly and permanently increase from ~0.8 pA to 2 pA.

Solution: If early model headstages are “hot swapped”, i.e. plugged in or unplugged while the instrument power is on, their headstage operational amplifiers might be damaged. Contact Sutter Technical Support for service.

Note: Best practices is to turn off the instrument power before (un)plugging its headstages.

7.3.4 Headstage Noise II

Problem: The noise levels of the instrument suddenly and erratically increase.

Solution: If the headstages are touched, the noise level will greatly increase. Make sure you are grounded or working in a Faraday cage.

7.3.5 Amplifier Output Attenuation

Problem: Axon amplifier analog output levels are attenuated when connected to a Sutter analog input channel.

Solution: The Axon analog output circuitry and the Sutter analog input circuitry interact to create a voltage divider, which attenuates the signal between them. Adjust for the 5% attenuation by scaling a routine input channel using a factor of “1.052632”.

7.3.6 Paradigm Sound Reduced

Problem: The paradigm ‘Sound’ step volume is attenuated at lower frequencies.

Solution: Upgrade the computer speaker, such as with add-on speakers.

7.3.7 Offset Zero Delay

Problem: The Offset button in the IPA Control Panel has a short delay before it responds.

Solution: This can occur after running the Membrane Test due to internal processing.

7.3.8 Metadata Written

Problem: Unchecking ‘Write’ in a metadata prompt still writes Experiment and Routine parameters.

Solution: A known issue. Contact Technical Support, or check for fixes in the SutterPatch Release Notes PDF file in the SutterPatch installer download: https://www.sutter.com/AMPLIFIERS/SutterPatch.html

7.3.9 Post-Acquisition Delay

Problem: Every time acquisition completes, there is a delay with the program operations.

Solution: The entire *.pxp Experiment file is resaved when a recording stops. Create new Experiments more often, so that file sizes are smaller and more manageable.

An optional strategy for streaming data acquisition that will not rewrite the data at the end of a recording is under development.

7.3.10 Sluggish Acquisition

Problem: Data acquisition is sluggish.

Solution: Increase the computer RAM, cache size or CPU speed, and/or upgrade the graphics system.

7.4 Analysis Issues

7.4.1 Analysis Not Deleted

Problem: An analysis cannot be deleted in the Analysis Editor.

Solution: The analysis is still in use, i.e. displayed in another window, such as a graph window - close the window to allow the analysis to be deleted.

7.4.2 Signal Axes Overlay

Problem: The X-axes and units are overlaid in the Scope window.

Solution: There is not enough room for the X-axes and units due to the number of signals displayed. Switch to a tiled signal layout, or reduce the number of visible signals by right-clicking a signal and selecting ‘Hide Signal’.

7.4.3 Graphs & Layouts Not Visible

Problem: Cannot see SutterPatch Experiment graphs or layouts on non-SutterPatch computers.

Solution: Use the Igor menu command Windows / Graph or Windows / Layout to see the object. Right-click it to modify with Igor options.

7.5 General Issues

7.5.1 Slow Computer

Problem: When using a slow computer in emulation (demo) acquisition mode, user actions can be difficult, such as clicking the Stop Acquisition button. You might need to click the button more than once, or hold it down longer than usual.

Solution: If computer resources are low, any ‘Live Preview’ options should be disabled. This option is found in the Routine Editor / Waveform Editor / Waveform / Sine – Squarewave - Chirp – Template dialogs. Using virtual input channels can also consume extra resources.

7.5.2 Slow Sweeps

Problem: When displaying a large number of sweeps, the display slows down.

Solution: Disable Persistence display in the Scope window.

7.5.3 Window Maximizing

Problem: Maximizing a window only maximizes the title bar.

Solution: Certain fixed-size windows and panels will not maximize (Dashboard, Paradigm Editor, Set Metadata, Set Preferences, Log, Action Potential Analysis, Event Detection.) This is a reported Igor issue.

7.5.4 Command Window Frozen

Problem: The Command Window is blank and/or unresponsive.

Solution: Use Ctrl-J, or click on the Amplifier Control Panel, and the Command window is redrawn as an active window. This is a reported Igor issue.

7.5.5 Strange Characters

Problem: Extra or strange characters appear in various fields when using Igor Pro 7.0.1.1.

Solution: Download and install the latest version of SutterPatch using the Full Installer available at https://www.sutter.com/AMPLIFIERS/SutterPatch.html.

7.5.6 Wrong Default Settings

Problem: Program preferences are non-standard or corrupted.

Solution: Reset the SutterPatch preferences to their defaults via the SutterPatch / Set Preferences / General / Preferences Defaults button.

7.5.7 Weird Behavior

Problem: There is weird or buggy behavior with the SutterPatch program.

Solution: Reset the SutterPatch program settings to their factory defaults via the SutterPatch / Set Preferences menu command, by performing a Factory Reset.

7.5.8 Igor Pro Features

Problem: There are a tremendous number of standard features in Igor Pro that can be used in conjunction with the SutterPatch application.

Solution: Please refer to the Igor Help browser, or to Wavemetrics, Inc., regarding issues with Igor Pro features.

APPENDIX A: LIMITED WARRANTY

§ Sutter Instrument Company, a division of Sutter Instrument Corporation, limits the warranty on this instrument to repair and replacement of defective components for two years from date of shipment, provided the instrument has been operated in accordance with the instructions outlined in this manual.

§ Abuse, misuse, or unauthorized repairs will void this warranty.

§ Warranty work will be performed only at the factory.

§ The cost of shipment both ways is paid for by Sutter Instrument Company during the first three months this warranty is in effect, after which the cost is the responsibility of the customer.

§ The limited warranty is as stated above and no implied or inferred liability for direct or consequential damages is intended.

§ An extended warranty for up to three additional years can be purchased at the time of ordering, or until the original warranty expires. For pricing and other information, please contact Sutter Instrument Company.

APPENDIX B: SOFTWARE LICENSE

SutterPatch Software Licensing Agreement

IMPORTANT NOTICE

PLEASE READ THIS CONTRACT CAREFULLY. BY USING ALL OR ANY PORTION OF THIS PROPRIETARY SOFTWARE YOU ACCEPT ALL THE TERMS AND CONDITIONS OF THIS AGREEMENT. YOU AGREE THAT THIS AGREEMENT IS ENFORCEABLE LIKE ANY WRITTEN NEGOTIATED AGREEMENT SIGNED BY YOU. IF YOU DO NOT AGREE, DO NOT USE THIS SOFTWARE.

1. DEFINITIONS.

Section 1.0. Defined Terms. Terms defined in this Article 1 shall have the meanings given below. Defined terms may be used in the singular or plural.

Section 1.1. “Agreement” means this Software License Agreement, which includes this Agreement’s terms and conditions, Schedules, Exhibits, Addenda, and Amendments, if any, which are incorporated in, and form an integral part of, this Agreement.

Section 1.2. “Confidential information” means any data or information, oral or written, of Sutter Instrument Corp., including without limitation, past, present, or future research, development or business activities, including any unannounced product(s) and service(s), and including any information relating to services, developments, inventions, processes, plans, financial information, customer lists, forecasts, and projections, including the terms of this Agreement.

Section 1.3. “End User” means the person or entity who is a client of Licensee and is authorized by User ID and Password to use the Product under Licensee’s authorization in the ordinary course of Licensee’s business.

Section 1.4. “Licensee” means a party or individual whose client has paid for and is using the Product or an End User, including any evaluation licensee.

Section 1.5. “Licensing Fees” mean the fees paid by a Licensee for the Product.

Section 1.6. “Product” means the software programs of Sutter Instrument Corp., including without limitation, SutterPatch software and corresponding documentation, printed materials and all updates or upgrades of the above that are provided to you, including without limitation, reports, graphs, test scores, interpretations and other information.

Section 1.7. “Term” means the length of time the License paid to use the Product (see current pricing schedule).

Section 1.8. “Website” means the Sutter Instrument Corp. website located at https://www.sutter.com and the Wavemetrics websites located at http://www.wavemetrics.com and https://www.igorpro.net.

2. GRANT OF LICENSE.

Section 2.1. Evaluation License. If the Product is used for evaluation, trial or demonstration purposes, Sutter Instrument Corp. grants such user a license solely for the purpose of evaluating, sampling, testing, or demonstrating the Product for the timeframe specified in the evaluation period. The evaluation license is subject to the following conditions: (i) Licensee hereby agrees to indemnify, defend and hold harmless Sutter Instrument Corp. and its officers, directors, employees and suppliers from and against any and all claims, damages, liabilities, costs and expenses, including without limitation, attorneys’ fees and costs, arising from or in any way related to the use of the Product, and (ii) otherwise comply with the terms of this agreement.

Section 2.2. Limited License. Subject to the terms and conditions set forth in this Agreement and payment of the license fee (see current pricing schedule), Licensor grants Licensee a non-exclusive, non-transferable, non-assignable license to access and use the Product during the Term. Licensee and Licensee’s permitted End Users shall be the only individuals permitted to use the Product, and Licensee is expressly prohibited from otherwise renting, leasing, loaning, selling or otherwise distributing its license rights.

Section 2.4. Description of Features and Functions. A complete description of the features and functions of the Product is available on the Website, and by accepting this Agreement, Licensee represents he/she has read and understands the nature, features, functions and limitations of the Product and agrees to the terms and conditions thereof.

3. USE OF PRODUCT.

Section 3.1. Licensee represents and warrants not to use or permit its End Users to use the Product to (i) upload, post or otherwise transmit any content that is unlawful, harmful, threatening, abusive, harassing, tortious, defamatory, vulgar, obscene, libelous, invasive of another’s privacy, hateful, or racially, ethnically or otherwise objectionable; (ii) harm minors in any way; (iii) impersonate any person or entity, falsely state or otherwise misrepresent his/her affiliation with a person or entity; (iv) forge headers or otherwise manipulate identifiers in order to disguise the origin of any content transmitted through the Service; (v) upload, post or otherwise transmit any content with no right to transmit under any law or under contractual or fiduciary relationships (such as inside information, proprietary and confidential information learned or disclosed as part of employment relationships or under nondisclosure agreements); (vi) upload, post or otherwise transmit any content that infringes any patent, trademark, trade secret, copyright or other proprietary rights of any party; (vii) upload, post or otherwise transmit any material that contains software viruses or any other computer code, files or programs designed to interrupt, destroy or limit the functionality of any computer software or hardware or telecommunications equipment; (viii) violate any applicable local, state, national or international law.

Section 3.2. Licensee agrees and shall cause each of his/her End Users to agree pursuant to a written agreement not to distribute, create, copy, duplicate, re-distribute, disassemble, decompile, reverse engineer, or sublicense the Product, and Licensee shall use best efforts to ensure the same. Licensee acknowledges, understands and agrees that Licensee shall acquire no rights or ownership interest whatsoever in the Product by its use of the Product, which ownership rights shall reside solely with Licensor or its affiliated companies.

Section 3.3. Licensee acknowledges and agrees that Licensee (and Licensee’s End Users) is solely and exclusively responsible for contents, data, information and communications that the Licensee and End Users upload and transmit through the Website to use the Product.

Section 3.4. Licensee agrees not to access the Product by any means other than was originally provided by Licensor for use when accessing the Product.

Section 3.5. As required by United States export regulations, Licensee shall not permit export of the Product, or any direct products thereof, to any country to which export is then controlled by the United States Bureau of Export Administration, unless Licensee has that agency’s prior written approval. Licensee shall require its End Users to comply with the provisions of this Section.

Section 3.6. If Licensee (or Licensee’s End User) is an office, branch or agency of the U.S. Government, then its use, duplication and disclosure of the Product or any part thereof, shall be subject to the following additional requirements: The Product is a “commercial item,” as such term is defined in 48 C.F.R. 12.101 (Oct. 1995), consisting of “commercial computer software” and “commercial computer software documentation,” as such terms are used in 48 C.F. 12.212 (September 1995). Consistent with 48 C.F.R. 12.212 and 48 C.F.R. 227.7202-1 through 227.7202-4, the U.S. Government End Users shall use the Product with only those rights set forth in this section.

Section 3.7. Licensor reserves the right to include in the Product one or more software devices to ensure that Licensee’s use of the Product are consistent with this Agreement.

Section 3.8. Licensee shall indemnify, defend and hold harmless Licensor and its officers, directors, suppliers and licensors of Licensor from any and all claims, damages, liability, costs and expenses, including without limitation, attorneys’ fees and costs arising from or in any way related to Licensee’s use or misuse of the Product and any other products purchased from Licensor, including without limitation, any derivative products of Licensee utilizing the Product, the hiring or firing of employees or consultants, or any business decisions made as a result of this Product.

Section 3.9. Licensee may not use, copy, modify, translate, or transfer the Product software, documentation, license key, or any of the files included with the Product software except as expressly defined in this agreement. Licensee may not attempt to unlock or bypass any copy-protection, licensing or authentication algorithm utilized by the Product software. Licensee may not remove or modify any copyright notice, nor any “About” dialog or the method by which it may be invoked.

4. FEES.

Section 4.1. Payment of the applicable Licensing Fee permits a Licensee to use the Product. Payment is due in full prior to or contemporaneously with the licensing or use of the Product. All Licensing Fees are payable in U.S. dollars and are non-refundable. Licensing fees may be collected as part of a bundled price included with other products from the Licensor.

Section 4.2. Where required by law, Licensor will collect from Licensee, and Licensee is responsible to pay for, sales and all other taxes associated with the order, unless Licensor is provided a valid and correct tax exemption certificate prior to order acceptance. The payment of such tax is the sole responsibility of Licensee.

5. SUPPORT.

Section 5.1. Licensor shall provide Licensee with a technical help desk facility to report problems or questions relating to the Product. The help desk is available by email at the following address:

info@sutter.com, or by calling the Licensor at 415-883-0128 between 8:00am and 5:00pm, Monday through Friday, Pacific Time. Licensor will usually reply within 48 hours of receipt of email.

6. INTELLECTUAL PROPERTY.

Section 6.1. Licensee recognizes and acknowledges the exclusive right of Licensor in and to all patents, trademarks, service marks, trade names, copyrights, and other intellectual property and proprietary rights in and to the Product and software, and that such patents, trademarks, service marks, trade names, copyrights, and other intellectual property and proprietary rights are the sole and exclusive property of Licensor

Section 6.2. Licensee waives its right to contest any of Licensor’s trademarks, service marks, trade names, copyrights, and other intellectual property and proprietary rights in and to the Product.

Section 6.3. Licensee shall not use such trademarks, service marks, and trade names except where permitted under this Agreement without receiving Licensor’s prior written approval of such use. If such approval is granted, Licensee’s right to use such trademarks, service marks, and trade names shall end upon the termination of this Agreement, unless otherwise notified by Licensor.

7. CONFIDENTIALITY.

Section 7.1. Licensee shall not disclose or otherwise make available the Product, including but not limited to, the physical media, reports, and documentation to any third party other than Licensee’s End Users. Unless otherwise agreed to by the parties, the scope of this Section shall be given the broadest interpretation possible and shall include, but not be limited to, the terms and conditions of this Agreement and pricing.

Section 7.2. The obligations of non-disclosure in Section 7.1 shall not apply to information that (i) is publicly available or in the public domain at the time disclosed; (ii) is, or becomes, publicly available or enters the public domain through no fault of the party receiving such information; (iii) is rightfully communicated to the recipient by persons not bound by confidentiality obligations with respect thereto; (iv) is already in the recipient’s possession free of any confidentiality obligations with respect thereto at the time of disclosure; (v) is independently developed by the recipient; (vi) is approved for release or disclosure by the disclosing party without restriction, (vii) is required by a court of competent jurisdiction.

Section 7.3. Licensee acknowledges that the Product software, in source code form, remains a confidential trade secret of Licensor and therefore Licensee agrees that it shall not modify, decompile, disassemble or reverse engineer the Software or attempt to do so. Licensee agrees to refrain from disclosing the Product software (and to take reasonable measures with its employees to ensure they do not disclose the Software) to any person, firm or entity except as expressly permitted herein.

8. TERMINATION.

Section 8.1 This Agreement shall become effective upon the use of the Product. Licensor may terminate this Agreement at any time upon the default by Licensee of any of the provisions of this Agreement with or without notice from Licensor to Licensee.

9. NO WARRANTIES.

Section 9.1. Although Licensor will make reasonable efforts to correct any material nonconformity between the Product and its then-current documentation that is brought to Licensor’s attention with written, documented reports from Licensee, LICENSOR DOES NOT AND CANNOT WARRANT THE RESULTS OBTAINED BY USING THE PRODUCT. Licensor’s entire liability and Licensee’s exclusive remedy under this Agreement shall be limited to the lesser of (i) the cost paid by Licensee for the software portion of the product, or (ii) $250.

Section 9.2. THE PRODUCT IS PROVIDED “AS IS.” LICENSOR SPECIFICALLY DISCLAIMS ANY AND ALL WARRANTIES TO LICENSEE OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, PERFORMANCE RESULTS AND NON INFRINGEMENT. THE ENTIRE RISK AS TO THE QUALITY AND PERFORMANCE OF THE PRODUCT IS WITH LICENSEE.

Section 9.3. IN NO EVENT WILL LICENSOR BE LIABLE TO LICENSEE FOR ANY DAMAGES, INCLUDING LOST PROFITS, LOST SAVINGS, OR OTHER INCIDENTAL OR CONSEQUENTIAL DAMAGES ARISING FROM THE USE OR INABILITY TO USE THE PRODUCT EVEN IF IT HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES, OR FOR ANY CLAIM BY ANY OTHER PARTY. Some states or jurisdictions do not allow the exclusion or limitation of incidental, consequential or special damages, or the exclusion of implied warranties, or limitations on how long an implied warranty may last, so the above limitations may not apply to Licensee. To the extent permissible, any implied warranties are limited to thirty (30) days. This warranty gives Licensee specific legal rights. Licensee may have other rights which vary from state to state or jurisdiction to jurisdiction.

Section 9.4. You acknowledge, understand and agree that your use of this Product is intended to complement your decision-making process and shall not be relied upon solely and completely to make business decisions but only in conjunction with your other business practices, policies, methods, research, investigations, and due diligence. Licensor’s license to use the Product is only provided to Licensee in full reliance upon Licensee’s representation and warranty to comply with this provision.

10. GENERAL PROVISIONS.

Section 10.1. Licensee warrants that Licensee has the requisite authority to enter into this Agreement.

Section 10.2. This Agreement constitutes the entire understanding and agreement between Licensor and Licensee regarding its subject matter, and supersedes all previous oral and written communications, agreements, memoranda, representations, or understandings between Licensor and Licensee regarding this Agreement. No other rights or licenses are granted to Licensee, except as expressly provided herein.

Section 10.3. Licensor may amend the terms of this Agreement and related company documents at any time with respect to any new releases, updates or versions of the Product, which if purchased by Licensee, will subject Licensee to the terms of the then current Licensor Software Licensing Agreement.

Section 10.4. This Agreement is not transferable or assignable by Licensee under any circumstances, without the prior written consent of Licensor. This agreement shall be binding upon, and is made for the benefit of, each party, its successors, and permitted assignees (if any). For the purposes of this Agreement, any change in control of Licensee shall constitute an assignment or transfer of this Agreement. As used in this section, a change in control is defined as (i) any change in ownership of more than fifty percent (50%) of the voting interest in Licensee, whether by merger, purchase, foreclosure of a security interest or other transaction, or (ii) a sale of all or substantially all of the assets of Licensee.

Section 10.5. The relationship established by this Agreement between Licensee and Licensor shall be that of licensee and licensor. Nothing contained in this Agreement shall be construed as creating a relationship of agency, joint venture or partnership between Licensee and Licensor, so that neither party shall have any right whatsoever to incur any liabilities or obligations on behalf of the other party.

Section 10.6. The laws of California shall govern this Agreement. Any action or proceeding brought by either party against the other arising out of, or related to, this Agreement shall be brought only in a state or federal court of competent jurisdiction located in California and the parties hereby consent to the personal jurisdiction of said courts.

Section 10.7. In the event that any provision of this Agreement is found invalid or unenforceable pursuant to a judicial decree or decision, the remainder of this Agreement shall remain valid and enforceable according to its terms.

Section 10.8. The headings provided in this Agreement are for convenience and reference purposes only. In the event of a conflict between the terms and conditions listed in Articles 1 through 10, and the attached Schedules, the terms and conditions shall govern.

Section 10.9. A waiver of a breach, violation, or default under this Agreement shall not be a waiver of any subsequent breach, violation or default. Failure of either party to enforce compliance with any term or condition of this Agreement shall not constitute a waiver of such term or condition.

Section 10.10. All notices and communications shall be in writing and shall be deemed to have been duly given when delivered or three (3) Business Days after mailing by certified mail, return receipt requested, postage prepaid, addressed to the parties at their respective addresses or at such other addresses as the parties may designate by written notice in accordance with this section.

Section 10.11. Any amendments or addenda to this Agreement, may be executed in counterparts, each of which will be considered an original, but all counterparts together will constitute one agreement. A facsimile of a signed copy of this Agreement, or an electronic or other digital signature imprinted on this Agreement, may be relied upon as an original.

Dimensions (in.)	1.375 (L) x 1 (W) x 0.8 (H)
Dimensions (cm)	3.5 (L) x 2.54 (W) x 2 (H)
Weight (lb)	0.21 0.294 (with cable)
Weight (kg)	0.095 0.133 (with cable)
Ground Socket	1 mm
Output Gain:	0 – 25x
Feedback Resistor	500 MΩ
Whole Cell Capacitance Compensation Range	0 – 100 pF
Current Clamp Rise Time	17.5 µs

IPA Headstage Noise (measured with 8-pole Bessel filter)
Bandwidth	Open-Circuit Noise (RMS)
0.1 – 1 kHz	< 0.25 pA
0.1 – 5 kHz	< 0.75 pA
0.1 – 10 kHz	< 1.40 pA

IPA Amplifier Bandwidth (Voltage Clamp)
Bandwidth	Availability
20 kHz	External (limited by Bessel filter)
50 kHz	Internal

IPA Data Acquisition
Analog Range (voltage)	±10 V
Analog Range (current)	±20 nA
Analog Resolution	16-bit
Maximum Input Frequency	20 kHz
Maximum Output Frequency	10 kHz
Digital Input Trigger Level	+2.3 V
Digital Output Trigger Level	+3.3 V, 100 µs

IPA Expansion Panel
Dimensions (in)	18.8 x 2 x 3.5
Dimensions (cm)	48 x 5 x 9
Weight (lbs)	3.5
Weight (kg)	1.6
Digital Output BNCs	8
Auxiliary Analog Input BNCs	4
Auxiliary Analog Output BNCs	2

IPA Electrical
Power consumption	18 Watts maximum
Mains fuse	250V 1A Slow Blow (5 mm x 20 mm) T2.0
Cables	Shielded grounded power line cord
Power Limits	88 VAC – 264 VAC


IPA Headstage
Construction:	Anodized aluminum case