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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. System Setup
- Construct the pressure control unit.
- Assemble the pressure control unit according to the circuit map (Figure 1). Solder the necessary parts onto the Printed Circuit Board (PCB) manufactured according to the electrical circuit schematics (Figure 1b). Use standard resistors, LEDs(Light-emitting diodes), Metal-Oxide Semiconductor field-effect transistors (MOSFETs), capacitors, and connectors. Solder solenoid valves onto the PCB (Printed circuit board). Connect the air pump and air pressure sensor to the PCB with an electrical wire.
NOTE: After all necessary parts are available, the pressure control unit should be constructed in about 2 hours.
- Connect the secondary data acquisition (DAQ) board.
- Connect data outputs from the printed circuit board to the DAQ board, following Table 1.
NOTE: The DAQ board will run in "Single-ended mode." The port map is in the user manual (see the Table of Materials).
- Connect "AIn Pr S" to one of the analog input (AI) channels and "R-Gr" to one of the analog grounds on the secondary DAQ board.
- Connect the primary output from the amplifier to one of the AI channels and the ground to the analog ground of the secondary DAQ board.
NOTE: A standard BNC cable can be used to connect the primary output from the amplifier.
- Strip the other end and connect the positive signal (i.e. copper core) to the AI channel and the ground (i.e. the thin wire around the core) to the analog ground. Repeat this step for a second channel if more than one patch channel is used.
NOTE: The analog input to the DAQ board will be configured in later steps.
- Connect power to the power output of the secondary DAQ board. Use a separate 12 V power source for the pump.
- Connect the tubing.
- Connect the air pump and the two valves.
- In the last step, use a 3-way connector to connect the soft tubing from the valve 2 top port to the pressure sensor and the pipette holder.
- If two pipettes are used, add another 3-way connector to the tubing connected to the pipette holder. When patching, manually switch between the valves and the pipettes in use.
- Install Autopatcher IG.
NOTE: System requirement: Autopatcher IG was only tested on a PC running Windows 7. It has not been validated for other operating systems. The described procedure applies specifically to the hardware listed in the Table of Materials.
- Download Autopatcher-IG from GitHub (https://github.com/chubykin/AutoPatcher_IG).
- Install Python (see the Table of Materials for the version and download address).
- Uninstall the PyQt4 library by typing "pip uninstall PyQt4" in a command line terminal.
NOTE: The system uses an older version of the PyQt4 library to achieve compatibility with the Qwt and Opencv libraries.
- Install Python libraries from historic wheel files (http://www.lfd.uci.edu/~gohlke/pythonlibs/). Find the following files: Numpy (pymc-2.3.6-cp27-cp27m-win32.whl), Opencv (opencv_python-2.4.13.2-cp27-cp27m-win32.whl), Pyqt (PyQt4-4.11.4-cp27-none-win32.whl), and Qwt (PyQwt-5.2.1-cp27-none-win32.whl).
- To install the wheel files, go to the directory where the files are saved and type "pip install ***wheelfilename***.whl." Substitute "***wheelfilename***" with the actual name of the file.
NOTE: "cp27" in the wheel file name indicates Python 2.7, and "win32" indicated Windows 32-bit. If "win32" does not work, try "win64."
- To control the CCD (charge-coupled device) camera, download and install the installer for 64-bit (https://www.qimaging.com/support/software/). Then download MicroManager for 64-bit (https://micro-manager.org/wiki/Download_Micro-Manager_Latest_Release) to control the camera in Python.
- To control the manipulators and the microscope stage, install control software provided by the manufacturer.
NOTE: By doing this, the driver necessary to control the manipulators is also installed. The installation package is commonly provided in a CD-ROM.
- To control the secondary DAQ board, install the Universal Library from CD-ROM, provided with the purchase of the DAQ board.
- Configure the hardware for Autopatcher IG.
- Connect the microscope stage and manipulator controllers to the computer via USB ports.
- Assign COM port numbers to unit 0: microscope stage, unit 1: left manipulator, and unit 2: right manipulator, in this order, in the "ports.csv" configuration file in the "configuration" folder. Leave the other parameters in the ports.csv file (i.e. "SCI" and "1") unchanged.
NOTE: The COM port number information can be found by running the manipulator configuration software provided by the manufacturer. Go to the "settings" tab, select "settings" and the "Motion" page, and read the labels for each tab at the top. Alternatively, this information can be found in the PC Device Manager.
- Assign analog input channel numbers on the DAQ board for a pressure sensor and patch channel 1 and 2 (corresponding to unit 1 and 2). Enter the channel number in the "DAQchannels.csv" file in the "configuration" folder.
NOTE: It is recommended to open the .csv files with the Notepad application instead of a spreadsheet, as it may alter the information when saving changes.
- Run Autopatcher IG.
- Turn on the amplifier, microscope controller, and manipulator controller. Ensure that the amplifier software is running.
- Run Autopatcher IG with Python from a command line terminal as follows: first, change the directory (command "cd" for most common terminals) where Autopatcher IG is installed, type "python Autopatcher_IG.pyw" in the command line terminal, and hit the "Enter" key.
NOTE: Do not run the manipulator control software before running Autopatcher IG because it will occupy the microscope stage and manipulator, causing Autopatcher IG to be unable to find the hardware. Manipulator control software can be run after Autopatcher IG is fully initiated if there are additional modules to be controlled (e.g., the inline heater).
- Calibrate the primary pipette.
- Pull patch pipettes. Fill a pulled glass pipette with an internal solution and load it onto the head stage.
NOTE: Empty glass pipettes have different contrasts under the microscope and may lead to inaccurate calibration.
- Move the pipette tip to the microscope visual field and bring it into focus. If the dial pad is used to move the manipulators and/or microscope stage, update the coordinates by pressing "z" on the keyboard.
NOTE: This action is not necessary if the keyboard (microscope stage: A/D - x-axis, W/S - y-axis, R/F - z-axis; manipulators: H/K - x-axis, U/J - y-axis, O/L - z-axis, 1/2 - unit number) is used to control movement because the coordinates will be updated in real-time.
- Click the "Start calibration" button on the main Graphic User Interface (GUI) for the corresponding unit on which the pipette is loaded (Figure 2).
NOTE: A pop-up window will appear when the calibration is finished.
NOTE: Calibration will be carried out automatically, which will take about 3.5 min. Clicking on the same button (switched now to "cancel calibration" after initiating calibration) will abort the calibration attempt.
- Save the calibration by clicking "save calibration" at the bottom of the main GUI (it saves the current calibration for both manipulators and can be loaded in the future).
NOTE: The field of view under low (4 or 10X) and high (40X) magnification must be aligned for secondary calibration to function properly. Please refer to the user manual of the optical system in use for the alignment procedures.
2. Automatic Patch Clamp Procedure
- Prepare acute brain slices
- Prepare glass pipettes for the patch clamp.
- Place one brain slice in the center of the recording chamber. Stabilize the slice with a slice hold-down or "harp."
- Detect the fluorescent cell.
- Find the area of interest under the 4X lens. Move the microscope stage by turning on click-to-move ("CTM") mode and clicking the center of the area of interest. Alternatively, use the keypad to move the microscope stage (A/D - x-axis, W/S - y-axis, R/F - z-axis).
- Switch to the high-magnification lens and adjust the focus by moving the microscope in the z-axis, using R/F on the keypad.
NOTE: It is recommended to adjust the water bath level so that the focal plane under the low- and high-magnification lenses are the same or similar in the z-axis.
- Click the "Detect Cell" button on the main GUI column, "Unit 0." If the LED or laser light source of the setup cannot be controlled by the TTL signal, manually turn on the LED/laser; a pop-up window will appear when the cell detection is finished.
- Turn off the LED/laser if necessary; a list of cell coordinates will appear in the "Memory positions" GUI. Remove undesired cells from the list by clicking the "X" button next to the coordinates.
- Alternatively, if target cells are not fluorescently labeled, click "Mouse mode" on the main GUI. Click on the cell of interest; a yellow dot with a number will appear on the cell, and the coordinates of the cell will appear in the "Memory positions" GUI.
- Calibrate the secondary pipette.
- Fill 1/3 of a glass pipette with internal solution. Load the pipette onto the pipette holder attached to the head stage.
- Use the low-magnification lens. Bring the pipette into the visual field and adjust the focus using the keypad (H/K - x-axis, U/J - y-axis, O/L - z-axis). Use "1" and "2" to switch between unit 1 and unit 2.
- Load the primary calibration by clicking on "Load calibration." Click the "Secondary calibration" button on the main GUI under the unit that is in use. For example, if unit 2 is in use, click the "Secondary calibration" button in the "Unit 2" column. Follow the pop-up window instructions to switch to the high-magnification lens.
- Patch a target cell.
- Make sure that the "MultiClamp" (i.e. amplifier) software is running. Click on the "Patch control" button to open the "Patch control" GUI; it may take up to a few min to open this GUI.
- Use the 40X magnification lens by checking "40X" on the main GUI "Unit 0" column. Click the "go to" button next to the cell of interest on the coordinate list in the "Memory position" GUI; the microscope will move to the cell.
- Click on the CTM button of the unit in use in the main GUI to enable movement following a mouse click. Click on the cell of interest; the pipette tip will move to the cell.
- Click on the "Patch" button on the "Patch control" GUI.
NOTE: Automatic patching will begin, and the pressure and resistance can be monitored on the "Patch control" GUI.
- Use the "Unit 1 selected" button to switch the input signal between the two units.
NOTE: The system will approach the target cell, apply negative pressure, match the cell membrane potential, and detect gigaseal formation based on a series of pressure and resistance thresholds and logic.
- Manipulate the automatic process at any point by clicking on the respective buttons on the "Patch control" GUI. For example, click on the "Patch" button again to cancel the patch trial and click on "Next stage" to advance the patching process to the next step, regardless of the threshold.
NOTE: A pop-up window will notify the user when a gigaseal has formed, and the option to apply zap along with large negative pressure will be presented.
- Select "Yes" to break in with combined zap and suction. Alternatively, select "No" to break in with suction only.
NOTE: When a successful whole-cell patch is completed, a pop-up window will remind the user to save the experiment patch log.
- Save the experiment patch log.
NOTE: If a patching trial is unsuccessful, a pop-up window will notify the user, and the patch process will reset.
- Go back to step 2.4 and repeat the steps with a different cell.