ANATOMICAL AND FUNCTIONAL IMAGING OF NEURONS USING 2-PHOTON LASER-SCANNING MICROSCOPY

Light scattering by brain tissue and phototoxicity are major obstacles to the use of high-resolution optical imaging and photo-activation (' uncaging') of bioactive compounds from inactive ('caged') precursors i n intact and semi-intact nervous systems. Optical methods based on 2-p hoton excitation promise to reduce these obstacles (Denk, 1994; Denk e t al., 1990, 1994). Here we show a range of imaging modes based on 2-p hoton laser scanning microscopy (TPLSM) as applicable to problems in n euroscience. Fluorescence images were taken of neurons labeled with io n-sensitive and voltage-sensitive dyes in invertebrate ganglia, mammal ian brain slices, and from the intact mammalian brain. Scanning photoc hemical images with whole-cell current detection (Denk, 1994) show how the distribution of neurotransmitter receptors on the surface of spec ific cells can be mapped. All images show strong optical sectioning an d usable images can be obtained at depths greater than 100 mu m below the surface of the preparation.