Three-dimensional reconstruction of cytoskeletons in rat salivary glands using confocal fluorescence microscopy and volume-rendering computer graphics]

A new reconstruction technique for the demonstration of three-dimensional architectural details from biological fluorescent specimens was used to determine the precise spacial organization of F-actin in rat sublingual glands. F-actin was stained with NBD-phallacidin in thick (20-30 microns) frozen sections and observed with a confocal laser scanning microscope to obtain thin (-1 micron) optically sectioned images. A series of optical sections taken successively at different focal levels in steps of 1 micron was then reconstructed with volume-rendering computer graphics. The rendered images viewed from several angles (e.g. top, bottom, sides) clearly revealed the presence of F-actin fluorescence along the hexagonal framework of the junctional complex within the acini and in the cytoplasm of star-shaped myoepithelial cells encircling the acini. The detailed structures and the topographical relations between the junctional complex and myoepithelial cells were more impressively observed when the rendered images were displayed by motion picture. These images are free from any artifacts caused by the mechanical sectioning and revealed well the delicate and complicated profiles of cellular structures that previously have been almost impossible to demonstrate by conventional reconstruction techniques. We expect that the combination of confocal microscopy and volume rendering will permit the dynamic observation of previously unseen phenomena in three dimensions, such as the behavior of biologically active molecules in living cells.