Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy

With a combination of infrared illumination, differential interference contrast (DIC) and image intensification by video, unstained living neurons have been visualized up to a depth of 50-100 microns in 300-microns thick brain slices. Recording and application pipettes could be placed under visual guidance. When applied to slices from rat neocortex and hippocampus, pyramidal and non-pyramidal neurons could be differentiated with medium magnification (20x-40x objective). Local neuronal clusters and bundling of apical dendrites of pyramidal cells were visible. The use of an objective with high numerical aperture (63x, N.A. 1.4) allowed the visualization of structures in the submicron range in neocortex and hippocampus. In combination with electrophysiological recordings, infrared DIC-videomicroscopy should facilitate the search for morphological changes underlying neuronal plasticity and the characterization of neuronal networks.