Optogenetic Electrophysiology Recording of Cells with Light-Dependent Ion Channels

Place a coverslip containing embryonic kidney cells expressing light-dependent ion channels and a fluorescent reporter protein on a silicon-applied measuring chamber. Close it and add an external buffer.

Position this chamber under a microscope. Place a bath electrode filled with agar. Perfuse with a buffer to remove residual media and dead cells.

Using a fluorescence filter, identify and focus on the target cell.

Attach a patch pipette to a micromanipulator. Apply pressure to prevent clogging and approach the cell.

Position the pipette near the cell and release positive pressure to form a membrane patch at the tip.

After forming the membrane patch, apply negative pressure to rupture the membrane.

Apply a light of a specific wavelength to open the ion channels. The movement of ions is recorded as voltage change by the electrode.

To begin, seal the measuring chamber with silicon to prevent leakage of the external buffer. Then, place one coverslip into the chamber and close it. Fill the chamber carefully with extracellular buffer to prevent the cells from detaching. Then, place the measuring chamber under the microscope using the 40x objective for cell visualization.

Next, put an aga