Confocal fluorescence microscopy with the tandem scanning light microscope

Applications of the tandem scanning confocal microscope (TSM) to fluorescence microscopy and its ability to resolve fluorescent biological structures are described. The TSM, in conjunction with a cooled charge-coupled device (cooled CCD) and conventional epifluorescence light source and filter sets, provided high-resolution, confocal data, so that different fluorescent cellular components were distinguished in three dimensions within the same cell. One of the unique features of the TSM is the ability to image fluorochromes excited by ultraviolet light (e.g. Hoechst, DAPI) in addition to fluorescein and rhodamine. Since the illumination is dim, photobleaching is insignificant and prolonged viewing of living specimens is possible. Series of optical sections taken in the Z-axis with the TSM were reproduced as stereo images and three-dimensional reconstructions. These data show that the TSM is potentially a powerful tool in fluorescence microscopy for determining three-dimensional relationships of complex structures within cells labeled with multiple fluorochromes.