Computer-controlled impalement of cells in retinal wholemounts visualized by infrared CCD imaging on an inverted microscope.

We present a computer-guided microelectrode positioning system that is routinely used in our laboratory for intracellular electrophysiology and functional staining of retinal neurons. Wholemount preparations of isolated retina are kept in a superfusion chamber on the stage of an inverted microscope. Cells and layers of the retina are visualized by Nomarski interference contrast using infrared light in combination with a CCD camera system. After five-point calibration has been performed the electrode can be guided to any point inside the calibrated volume without moving the retina. Electrode deviations from target cells can be corrected by the software further improving the precision of this system. The good visibility of cells avoids prelabeling with fluorescent dyes and makes it possible to work under completely dark adapted conditions.