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