This ppt walks you through the basics of PATCHMASTER software: how to get started.

1. Using PATCHMASTER in
Standard Patch Clamp Experiments
Live Demonstration for Beginners
BioPhysical Society – 61st Meeting
New Orleans, February 11th to 15th
Speaker: Frank P. Elsen, Ph.D.
Electrophysiology Application Scientist

2. 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

3. PATCHMASTER – Simple Handling & Easy Work Flow
Protocol Editor
Manages & controls entire experiment
Automatic
control
Parameters: Input /
Readout
Information: Readout
Oscilloscope
Displays data
live & recorded
Notebook
Keeps written
record of entire
experiment
Replay
Displays data
live & recorded
• Analysis display
• Parameters
Scientists
MANUAL CONTROL
Amplifier
Controls Patch-
Clamp amplifier
Pulse
Generator
Creates stimulus
files and sets DA
and AD channels
Online
Analysis
Online analysis of
live data recording
Control
Easy access to
experiment
controls & info
• Configuration
• I/O Control
• Extensions
• Solution Base / Changer
P I

4. Installation Procedure
 Hardware Installation: Plug-in EPC10_USB to direct USB port (PC or Mac)
 Download (www.heka.com) & install driver for protection key (dongle)
 Download & install PATCHMASTER
• Run “Installer.exe” as administrator
• 1st time run “PATCHMASTER” as administrator, afterwards not required
Illuminated LED
indicates “fully functional”
C:Program Files (x86)HEKAPatchMaster
(Example: PC)

5. PATCHMASTER – Graphical User Interface
 Different windows for different functions
 Fully customizable and modifications are automatically saved in the PATCHMASTER.set file (Save & Exit)
Amplifier
- Control & Set
Oscilloscope
- Display data (live & stored)
Replay
- Easy data access
Notebook
- Create written record
Control
- Easy access to applications
Pulse Generator
- Create Stim. Sequences
“Windows” menu

6. Save Your Settings – “Patchmaster.set”
 PATCHMASTER automatically starts with the settings determined by 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. How to save the “Patchmaster.set” file
2. How to create user-specific “Patchmaster.set” files
3. How to change colors & texts using dia-files (optional)
4. Common settings to be made in the “Configuration window”
5. How to create user-specific short-cut to start PATCHMASTER
To open configuration dialogue:
Ctrl (hold) & left-click on box

7. 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
1. Create a “New Experiment” data file (Root-level)
2. Optional: Open an existing data file to add data to it
3. Add text to the “Root” and/or “Group”
4. Change the label of the “Group” (Cell)
5. We recommend to create a “New Group” for each new cell

8. Establish Recording Configuration
 Use “manual” or “automatic” controls in the Amplifier window
 Use the “Protocol Editor” to fine-tune the automatic controls (AUTO-Buttons)
1. Compensate “Offsets”
1. Switch ON test pulse, adjust test pulse settings
2. Apply constant positive pressure to pipette solution
3. Lower pipette into bath, close to cell
4. Set Liquid-liquid-junction-potential (L J) & press “Auto” offset button (1)
2. Establish seal & compensate pipette capacitance
1. Lower pipette until it touches cell membrane
2. Reverse pipette pressure and observe “test pulse” and “resistance value”
3. Apply holding potential (V-memb) of -60 mV (optional)
4. 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
1. Observe “test pulse” & apply rapid suction pulses
2. STOP pressure adjustments immediately, when cell capacitance transient appears
3. Set “Range” (estimated max. cell size) & press “Auto” C-slow button (3)
4. To compensate “Serial Resistance (Rs)”, set compensation speed & press “Auto” (4)
5. Adjust percentage of error compensation as needed
6. Switch OFF test pulse – ready for experiments
Manual Approach

9. Establish Recording Configuration
 Use “manual” or “automatic” controls in the Amplifier window
 Use the “Protocol Editor” to fine-tune the automatic controls (AUTO-Buttons)
1. Compensate “Offsets”
1. Apply constant positive pressure to pipette solution
2. Lower pipette into bath, close to cell
3. Press “SETUP” (1) or “OPEN-Pip”
2. Establish seal & compensate pipette capacitance
1. Lower pipette until it touches cell membrane
2. Reverse pipette pressure and observe “test pulse” and “resistance value”
3. After “seal resistance” reaches 1 – 10 GW – press “SEAL” (2) or “CAP”
3. Establish Whole-cell recording configuration
& compensate cell capacitance and cell input resistance
1. Observe “test pulse” & apply rapid suction pulses
2. STOP pressure adjustments immediately, when cell capacitance transient appears
3. Press “WHOLE-CELL” (3)
4. Ready for experiment
Using SETUP, SEAL & WHOLE-CELL buttons

10. Adjust (fine-tune) AUTO-Buttons
 Please note: This procedure is optional and shown here only for test purpose
 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)
• 3: Sets liquid-liquid-junction potential, if it is always the same.
Otherwise set it manually, before pressing “OPEN-Pip”.
• 4: Pressing the “Auto-offset” button
• 5-8: Switching ON & setting parameters of the Test Pulse
2. CAP (SEAL) (Cell-Attached-Patch)
• 1: Pressing the “Auto” button of C-fast
• 2: Sets “V-memb” to “-60 mV”
3. WHOLE-CELL
• 2: Sets appropriate cell size or remove this command line and set
it manually, before pressing “WHOLE-CELL”
• 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

11. 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, while acquiring data
• 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 sample time in [s] (ex. 0.0001) or left-click-hold
& scroll)
• Enter “Sweep Interval” 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
• To disable “Online Analysis”, open the “Analysis” window and select
“No Analysis”

12. 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, while acquiring data
• “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 the “Store” box (2)
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)

13. 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, while acquiring data
• 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)
2. Select which trace to display in “Trace Properties”
• Uncheck “Show” to NOT display specified trace

14. Analyzing Data
 Analyze (I-V-curve) data traces while recording them
 Use “Analysis” window to manage online and offline analysis
 Display “Analysis Results” using two separate “Analysis Display” windows
 Create new “Analysis Method”
• 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” to enable an analysis method determined
by the link from the pgf-sequence
• “Pool” will use the specified method from the analysis file
• “Assigned” will use the specified method from the data bundle
• 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

15. Analyzing Data
 Analyze (I-V-curve) data traces while recording them
 Use “Analysis” window to manage online and offline analysis
 Display “Analysis Results” using two separate “Analysis Display” windows
• Open “Analysis Display” window by selecting it from the main
“Windows” menu
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

16. Acquiring Data
 Create new stimulation sequence (Ramp) using “Pulse Generator” window
 Create new analysis method using “Analysis” window & link them together
• Open “Pulse Generator” (press F8)
1. Set SWEEP & TIMING parameters
• Set number of sweeps to “1”
• Set acquisition frequency to “0.0001” (or left-click-hold & scroll)
2. Set DA (stimulation) and AD (recording) channels
• Set “DA” to “Stim-DA” and “StimScale”
• Set 1st AD to “Imon2” and 2nd AD to “Vmon”
• Check “Store” boxes of both channels
3. Enter values for SEGMENTS
• Select “Stored” to activate a segment
• Set first and 5th to “Constant” with “holding” and “10 ms”
• Set 2nd to “Constant” with “-80” Voltage and “2 ms” duration
• Set 3rd to “Ramp” with “20” Voltage and “100 ms” duration
• Set 4th 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)

17. 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 1st “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”

18. 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

19. Acquiring Data - Metadata
 Metadata are being saved with the data in the data-file
 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 again by
selecting data traces 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

20. 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)
1. Select the next free box & enter “Multi-Ramp” in
name pop-up window
2. With the first command line highlighted, press “Insert
After” and select “Repeat” from the pop-up window
3. Enter “5” in the “No. of Repeats” box
4. With the 1st command line highlighted, press “Insert
After” and select “Amplifier”, check “C-Slow” box
(executes “Auto-C-Slow”)
5. With the 2nd command line highlighted, press “Insert
After” and select “Wait” & enter “2” in the duration
box (to allow “Auto-C-Slow” to safely finish)
6. With the 3rd command line highlighted, press “Insert
After” and select “Acquire Series” & press “Select
PGF-Template”
7. From the pop-up window select “Ramp”
2. Run “Multi-Ramp” protocol

21. Exporting Data
 Exporting data traces with “Copy & Paste” or “Export as wmf-file”
 Export analysis values from the “NOTEBOOK” in ASCII, Igor or MatLab format

22. Exporting Data
 Exporting data traces with “Copy & Paste” or “Export as wmf-file”
 Export analysis values from the “NOTEBOOK” in ASCII, Igor or MatLab format