Automated Image-Guided Patch Clamp Technique for Studying Neurons in Brain Slices

Begin with an immobilized brain slice in a recording chamber.

Using the camera-controlled software of a microscope, focus on the target area

Turn on the excitation light and take multiple pictures at different depths.

The software detects the coordinates of fluorescently labeled cells.

Now, position the solution-filled patch pipette containing the recording electrode into the chamber.

Use suitable software to calibrate the position controls.

Under high magnification, recalibrate the controls to precisely target the fluorescently labeled cell.

Select the coordinates of the target cell.

Start the automatic lowering of the patch pipette.

The computer monitors the resistance via the electrode. Apply a negative pressur, an increase in resistance due to the pipette's contact with the cell membrane indicates gigaseal formation.

Following this, the software further decreases the pressure to puncture the cell membrane and obtain a whole-cell patch configuration.

In this step, place one brain slice in the center of the recording chamber. Stabilize the brain slice with a slice holdown or a harp. To detect the fluorescent cell, find the area of interest under the four times magnification. Then, move the microscope stage by turning on "click-to-move" mode, and click the center of the area of interest. Next, switch to the high magnification lens and adjust the focus by moving the microscope in the z-axis using RF on the keypad.

The software directs the microscope in the camera to take a series of images at different depths. Then, these images are subjected to computer vision processing to find fluorescently labeled cells. Click the Detect Cell button on the main column, unit zero. A list of cell coordinates will appear in the memory position's GUI. Remove undesired cells from the list by clicking the X button next to the coordinates.

Then, 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 position's GUI. To perform secondary calibration in order to detect pipette offset coordinates, fill one-third of a glass pipette with internal solution. Then, load the pipette onto the pipette holder attached to the head stage.

At low magnification, use one and two on the keypad to switch between unit one and unit two. Then, bring the pipette into the visual field and adjust the focus using the keypad.

Next, load the primary calibration by clicking on Load Calibration. Switch the microscope lens to high magnification, and click 40 times on the main GUI. Bring the pipette tip to the center. Then, click the secondary calibration button on the main GUI under the unit that is in use. To patch a target cell, click on the Patch Control button to open the patch control GUI. Click the Go To button next to the cell of interest on the coordinate list in the memory position GUI.

Subsequently, click on the CTM button of the unit in the main GUI to enable movement. Click on the cell of interest to move the pipette tip to the cell.

Next, use the Unit 1 Selected button to switch the input signal between the two units. Click on the Patch button on the patch control GUI. Automatic patching will begin, and the pressure and resistance can be monitored on the patch control GUI.

The automatic patching algorithm monitors resistance and controls pressure through a series of logic loops to form a gigaseal. A pop-up window notifies the formation of a gigaseal.

The system utilizes resistance change to recognize cell surface contact. In a situation that the cell surface contact is not detected in time, use the Next button to advance patching stage while remaining in the same automatic patching trial.

Manipulate the automatic process at any point by clicking on the respective buttons on the patch control GUI. Then, select Yes to break in with combined zap and suction. Then, save the experiment patch log.