Using PATCHMASTER in Standard Patch-Clamp Experiments

Transcription

1 Using PATCHMASTER in Standard Patch-Clamp Experiments Live Demonstration for Beginners BioPhysical Society 61 st Meeting New Orleans, February 11 th to 15 th, 2017 Frank Elsen, Ph.D. Electrophysiology Application Scientist Overview PATCHMASTER Simple Handling & Easy Work Flow Installation Procedure (PATCHMASTER & EPC10_USB) PATCHMASTER o Graphical User Interface (GUI) o Save Your Settings (Patchmaster.set) o Start a New Experiment o Establish Recording Configuration o Adjust (fine-tune) AUTO-Buttons o Acquiring Data (create & start pgf-sequence, adjust trace display) o Analyzing Data (create & link online analysis) o Use Protocols to run complex experiments o Export data traces and data values 1

2 PATCHMASTER Simple Handling & Easy Work Flow Scientists MANUAL CONTROL Automatic control Protocol Editor Manages & controls entire experiment P Parameters: Input / Readout Information: Readout I Amplifier Controls Patch- Clamp amplifier Pulse Generator Creates stimulus files and sets DA and AD channels Oscilloscope Displays data live & recorded Notebook Keeps written record of entire experiment Control Easy access to experiment controls & info Online Analysis Online analysis of live data recording Analysis display Parameters Replay Displays data live & recorded Configuration I/O Control Extensions Solution Base / Changer Installation Procedure Hardware Installation: Plug-in EPC10_USB to direct USB port (PC or Mac) Download ( & install driver for protection key (dongle) Download & install PATCHMASTER Run Installer.exe as administrator 1 st time run PATCHMASTER as administrator, afterwards not required C:\\Program Files (x86)\heka\patchmaster Illuminated LED indicates fully functional (Example: PC) 2

3 PATCHMASTER Graphical User Interface Different windows for different functions Fully customizable and modifications are automatically saved in the PATCHMASTER.set file (Save & Exit) Windows menu Amplifier - Control & Set Replay - Easy data access Oscilloscope - Display data (live & stored) Notebook - Create written record Control - Easy access to applications Pulse Generator - Create Stim. Sequences Save Your Settings Patchmaster.set PATCHMASTER automatically starts with the settings of the Patchmaster.set file Rename or delete Patchmaster.set to start PATCHMASTER with its default settings Each user should create their own Patchmaster.set file and short-cut to start PATCHMASTER 1. Save your settings in your Patchmaster.set file Press SAVE or SAVE AS in the Configuration window Press Save and Exit when closing PATCHMASTER 2. Change colors & texts using dia-files (optional) Changing text of AUTO-buttons (ex.: from SEAL to Open-Pip ) Recommendation: Create user-specific short-cut to start PATCHMASTER with your personalized Patchmaster.set file To open configuration dialogue: Ctrl (hold) & left-click on box that you want to modify Enable to activate feature 3

4 Create your own short-cut to start PATCHMASTER If multiple researchers use the same setup, every user can create their own PATCHMASTER configuration file (Patchmaster.set) and start it with their own shortcut. 1. First each user has to create their own Patchmaster.set file: Start PATCHMASTER and adjust all settings to your liking (window placement & size, file pathways, hardware configurations, etc.) Open Configuration window and select SAVE AS, chose a user specific name for the set-file (ex. Patchmaster_user_name.set) Save this new set file into the Patchmaster folder (same location as the original set-file) 2. Create a user specific shortcut to start PATCHMASTER Close PATCHMASTER Create a shortcut for the Patchmaster.exe file on the desktop, right-click on this shortcut, select Properties, click on Shortcut tab and highlight the entire target path that is inside the target box Copy & paste the entire target path behind the original target path, so that two identical pathways are now present in the target box Important: Make sure that there is a space (red arrow) between the two pathways Please see the example below: "C:\Program Files (x86)\heka\patchmaster\patchmaster.exe" "C:\Program Files (x86)\heka\patchmaster\patchmaster.exe" Replace the second PatchMaster.exe with the respective Patchmaster_user_name.set Rename the shortcut to Patchmaster_user 3. Using this shortcut will start PATCHMASTER with the user specific set-file and any changes made will be saved to that specific set-file Start a New Experiment Create a new data file or use an old data file and add data to it Use label & text fields to add metadata information to the data file Open Replay window (press F10) (recommended to leave it open at all times) 1. Create new or open existing data file Recommended to create new data file for every day of experiments Alternatively, you can use the same data file for one project (use Open Modify ) 2. Use New Group and/or New Experiment to keep data nicely organized (just a recommendation) Use New Group for each new cell Use New Experiment only, if data will belong to a different project 3. Enter Text & edit Label Text can be added to: Root, Group and Series Label can be edited of: Group, Series, Sweeps and Traces Add as much text as needed, better too much than too little We recommend that you still create a hand-written log for your experiments 4

5 Establish Recording Configuration Use manual or automatic controls in the Amplifier window Use the Protocol Editor to fine-tune the automatic controls (AUTO-Buttons) Manual Amplifier Control 1. Compensate Offsets Switch ON test pulse, adjust test pulse settings Apply constant positive pressure to pipette solution Lower pipette into bath, close to cell Set Liquid-liquid-junction-potential (L J) & press Auto offset button (1) 2. Establish seal & compensate pipette capacitance Lower pipette until it touches cell membrane Reverse pipette pressure and observe test pulse and resistance value Apply holding potential (V-memb) of -60 mv (optional) After seal resistance reaches 1 10 GW press Auto C-fast button (2) 3. Establish Whole-cell recording configuration & compensate cell capacitance and cell input resistance Observe test pulse & apply rapid suction pulses STOP pressure variations immediately, when cell capacitance transient appears Set Range (estimated max. cell size) & press Auto C-slow button (3) To compensate Serial Resistance (Rs), set compensation speed & press Auto (4) Adjust percentage of error compensation as needed Switch OFF test pulse (EPC10_USB-2 double) Ready for experiments Establish Recording Configuration Use manual or automatic controls in the Amplifier window Use the Protocol Editor to fine-tune the automatic controls (AUTO-Buttons) (EPC10_USB-2 double) Using SETUP, SEAL & WHOLE-CELL buttons 1. Compensate Offsets Apply constant positive pressure to pipette solution Lower pipette into bath, close to cell Press SETUP The 1 st AUTO-button can also be engaged by pressing number pad key 1 2. Establish seal & compensate pipette capacitance Lower pipette until it touches cell membrane Reverse pipette pressure and observe test pulse and resistance value After seal resistance reaches 1 10 GW press SEAL The 2 nd AUTO-button can also be engaged by pressing number pad key 2 3. Establish Whole-cell recording configuration & compensate cell capacitance and cell input resistance Observe test pulse & apply rapid suction pulses STOP pressure adjustments immediately, when cell capacitance transient appears Press WHOLE-CELL The 3 rd AUTO-button can also be engaged by pressing number pad key 3 Ready for experiment 5

6 Adjust (fine-tune) AUTO-Buttons Please note: This procedure is optional Use the Protocol Editor to fine-tune the automatic controls (AUTO-Buttons) Open Protocol Editor (press F9) Load/Save user-specific pro-file (try not to modify default files) 1. OPEN-Pip (SETUP, 1 st AUTO-button) 3: Sets liquid-liquid-junction potential, if it is always the same. Otherwise set it manually, before pressing OPEN-Pip. 4: Engaging the Auto-offset button 5-8: Switching ON & setting parameters of the Test Pulse 2. CAP (SEAL, 2 nd AUTO-button) 1: Engaging the Auto button of C-fast 2: Sets V-memb to -60 mv 3. WHOLE-CELL (3 rd AUTO-button) 2: Sets appropriate cell size, before engaging WHOLE-CELL (3) (option: remove this command line and set it manually) 4-5: Sets and executes Rs-compensation 6: Waits (halts) protocol for 2 s, to allow Rs-comp to finish 7: Switches OFF Test Pulse Ready for the experiment Acquiring Data Edit existing stimulation sequence (IV-steps) using Pulse Generator window Use Control window to manage experiment Easily switch between recording (saving) data and just evaluating (not saving) a data trace Adjust Oscilloscope settings Open Pulse Generator (press F8) Load/Save user-specific pgf-file (try not to modify default files) 1. Set SWEEP & TIMING parameters Set number of sweeps (ex. 12) Set acquisition frequency (enter time in [s] (ex ) or click-hold & scroll) Optional: Enter Sweep Interval if you want to wait for a given time between sweeps 2. Set DA (stimulation) and AD (recording) channels Left-click to open drop-down windows & select desired channels Check Store box to enable saving-option 3. Enter values for SEGMENTS To add a segment, left-click top box (next to segment #) Select desired Segment Class & set holding amplitude (ex. #2: -80) Set desired duration (in ms ) for each segment Complete any other desired settings & check cartoon drawing Optional: To disable Online Analysis, open the Analysis window and select No Analysis or (4) delete entry in Analysis box (IV) 6

7 Acquiring Data Edit existing stimulation sequence (IV-steps) using Pulse Generator window Use Control window to manage experiment Easily switch between recording (saving) data and just evaluating (not saving) a data trace Adjust Oscilloscope settings Control window should always be open 1. Start PGF-SEQUENCE or PROTOCOL Left-click on PGF or Protocol box starts selection immediately Toggle between saving data (red) and not-saving (pink) by pressing (2) the Store box 2. Controls to run the experiment Left-click on box to execute its function immediately Break stops sweep immediately, Stop interrupts at end of sweep Protocol: Next stops Series at end of sweep and next protocol event is executed 3. Status display of running experiment Data recording (acquisition) indicated by left box Time of the day (24 hours) & total PATCHMASTER running time (press Set to reset) Acquiring Data Edit existing stimulation sequence (IV-steps) using Pulse Generator window Use Control window to manage experiment Easily switch between recording (saving) data and just evaluating (not saving) a data trace Adjust Oscilloscope settings The Oscilloscope window should always be open 1. Adjust position of traces & set Digital Filter Select trace that you want to adjust & press Repaint to display all sweeps (make sure Series is selected in the Replay window) Left-click-hold & scroll Y-offs. to move selected trace to desired position, then press Repaint (click on X to center offset) Left-click-hold & scroll Y-scale to resize selected trace, then press Repaint (click on R to reset scale & offset) Press Auto Swp or Auto Ser to auto-scale selected trace(s) Left-click-hold & scroll Dig. Filter to select digital filter settings, please note trace changes while scrolling (or enter value in Hz) To evaluate traces, use Measure or Scan buttons 2. Selection in Replay window determines what is being displayed Selection will be displayed with all subcategories Selecting a Trace, only this trace will be displayed 3. Select what Traces from a Series to display Open Trace Properties from the Display main menu Uncheck Show to NOT display any of the specified traces 7

8 Analyzing Data Analyze (I-V-curve) data traces while recording them Use Analysis window to manage online and offline analysis Display Analysis Results using the Analysis Display windows Open Analysis window by pressing F7 1. Enable analysis, link to PGF-sequence & set analysis functions Select No Analysis to switch OFF any analysis functionality Select Use selected method to enable the currently active method Select Auto Stim Control - Pool to enable an analysis method determined by the link from the pgf-sequence To select an analysis function, click on Change 2. Set parameters of analysis graph Switch to ON to display graph & edit parameters Check box to select Analysis display window for graph 3. Replay data to execute analysis Analyzing Data Analyze (I-V-curve) data traces while recording them Use Analysis window to manage online and offline analysis Display Analysis Results in the Analysis Display windows Open Analysis Display window by selecting it from the main Windows menu (either Analysis Window 1 or 2) 1. Data displayed as graph or measured values Graphs can be displayed in two separated Analysis Display windows Analysis results can also be displayed as values in the notebook 8

9 Acquiring Data Create new stimulation sequence (Ramp) using Pulse Generator window Create new analysis method using Analysis window & run it with the Ramp-sequence Open Pulse Generator (press F8) 1. Set SWEEP & TIMING parameters Set number of sweeps to 1 Set acquisition frequency to (or left-click-hold & scroll) 2. Set DA (stimulation) and AD (recording) channels Set DA to Stim-DA and StimScale Set 1 st AD to Imon2 and 2 nd AD to Vmon Check Store boxes of both channels 3. Enter values for SEGMENTS Select Stored to activate a segment Set first and 5 th to Constant with holding and 10 ms Set 2 nd to Constant with -80 Voltage and 2 ms duration Set 3 rd to Ramp with 20 Voltage and 100 ms duration Set 4 th to Constant with 20 Voltage and 2 ms duration 4. Enable & specify link to online analysis Enter name of Analysis Method to create a link to analysis window Specify relevant segment for analysis (X and Y) Acquiring Data Create new stimulation sequence (Ramp) using Pulse Generator window Create new analysis method using Analysis window & run it with the Ramp-sequence Open Analysis window by pressing F7 1. Enable analysis, link to PGF-sequence & set analysis functions Select Auto Stim Control: Pool to enable the Ramp IV analysis method as being determined by the link from the Ramp -sequence Select box 2 of the Analysis Pool & enter Ramp IV Box 1 of the Analysis Functions is automatically selected Press Change to open Function Type window and select Stimulus In the name box enter Voltage [mv] Set Cursors Relative to Segment and check Ramps only box Select box 2 of the Analysis Functions, press Change select Trace In the name box enter Current [pa] & check Ramps only box 2. Set parameters of analysis graph Switch to ON to display graph & check 1 st Graph Entries box Select Voltage for X and Current for Y as Graph Entries Set X to Min -90m and Max 30m, set Y to -180p and 40p Graph Positions: Check box 1 for Graph in Window 1 9

10 Acquiring Data Create new stimulation sequence (Ramp) using Pulse Generator window Create new analysis method using Analysis window & run it with the Ramp-sequence Open Analysis Window 1 (display), select it from Windows menu Run PGF-sequence by pressing Ramp in the Control window 1. Data display in OSSCILOSCOPE window Adjust scaling and offset for Trace 1 (Current response, Imon2) 2. Display of analysis results from analysis method (Ramp IV) Analysis result (I-V relationship) is being displayed in the Analysis Window 1 while PGF-sequence Ramp is executed Metadata are being saved with the data in the data-file Acquiring Data - Metadata Metadata can be accessed during offline analysis from the data file Open Parameters window by selecting it from Windows menu Different metadata parameters can be accessed from different tabs Parameters window values are saved with each data sweep and can be accessed by selecting the data trace in the Replay window 1. Amplifier Items (Amplifier settings & values) All crucial amplifier settings and parameters are saved here V-offset, C-fast, C-slow and Rs values are being updated every time the respective Auto button is pressed 10

11 Acquiring Data - Protocol Create new protocol (Multi-Ramp) using Protocol Editor window Run new protocol (Multi-Ramp) Open Protocol Editor window by pressing F9 1. Create new protocol (Multi-Ramp) Select the next free box & enter Multi-Ramp in name pop-up window With the first command line highlighted, press Insert After and select Repeat from the pop-up window Enter 5 in the No. of Repeats box With the 1 st command line highlighted, press Insert After and select Amplifier, check C-Slow box (executes Auto-C-Slow ) With the 2 nd command line highlighted, press Insert After and select Wait & enter 2 in the duration box (to allow Auto-C-Slow to safely finish) With the 3 rd command line highlighted, press Insert After and select Acquire Series & press Select PGF-Template From the pop-up window select Ramp 2. Run Multi-Ramp protocol Exporting Data Exporting data traces with Copy & Paste or different file formats Export analysis values from Graphs or Notebook 1. Copy & Paste OSCILLOSCOPE display Adjust Oscilloscope display to your liking and have the Oscilloscope window in the foreground To copy the display to the clipboard you can either: o Press & Hold Ctrl & press C o Select Copy from Edit main menu Paste the clipboard to any suitable program 2. Export displayed traces in different formats Select format (ASCII, IgorPro, MatLab, wmf, etc.) Modify export settings depending on format Two options to export the display: o Press Export to export the display exactly as you see it o Press Export Full Sweep to export entire sweep, no matter what is displayed 3. Export & Import Traces only Select the Trace that you would like to use in the REPLAY window Select the desired format 11

12 Exporting Data Exporting data traces with Copy & Paste or different file formats Export analysis values from Graphs or Notebook REPLAY Export Format Data trace used for tail current analysis First select Export Format, then specify what to export by selecting the respective Export Mode Direct export into known formats, like IgorPro, MatLab or ASCCI are possible REPLAY Export Mode REPLAY Export Mode 1. Export Analysis Window 1 Here we export the values as ASCII-file that can be easily imported in EXCEL or GraphPad Prism Analysis results are displayed in an Analysis Window Select ASCII from Replay Export Format Select Analysis Graphs from Replay Export Mode Select Export Full Sweep to export entire sweep 2. Export Notebook Now we want to export the same analysis values from the notebook, keep ASCII as Export Format Select Analysis Notebook from Replay Export Mode Select Export Full Sweep to export entire sweep You can always save the Notebook as a text-file Notebook HEKA Support Team Martin Oberhofer, Ph.D. Technical Support Manager Central Europe, HEKA/Germany Frank Elsen, Ph.D. Electrophysiology Application Scientist, HEKA/USA Supporting the Support Team: o o o HEKA/Germany: Production/workshop facilities, Hardware and Software Engineers WARNER INSTRUMENTS distribution center & workshop Sales & Marketing HEKA/Germany and HEKA/USA Please feel free to send your inquiries to: support&heka.com 12