Backscattered electron imaging. Its application to biological specimens stained with heavy metals.

The present study demonstrates the applicability of backscattered electron images to the scanning electron microscope (SEM) observation of various biological specimens stained with heavy metals. Techniques of specimen preparation for backscatter imaging were introduced and results obtained by several staining methods were shown: silver staining for argyrophile fibers (Ishii's modification of Bielschowsky-Maresch's stain), Golgi staining for intracellular canaliculi of parietal cells, osmium-staining of intracellular lipid droplets, periodic acid-thiosemicarbazide-osmium staining for the detection of periodate-reactive mucosubstances and polysaccharides, and enzyme immunohistochemistry for bioactive peptides. Observation of stereo-pairs of backscatter images augments information on the three-dimensional localization of stained substances. Moreover, pairs of secondary emission and backscatter images of the same area clearly visualize the correlation between the surface structure and stained substances. Colored images by superimposition of secondary emission and backscatter information are also useful for an understanding of the orientation of the stained substances in three-dimensional structures. It is suggested that backscatter imaging can be widely used for various topochemical studies in three-dimensional extensions of biological specimens.