A novel method for viewing heavy metal stained and embedded biological tissue by field emission scanning electron microscopy

Abstract

Backscattered electron (BSE) imaging was used to display heavy metal stained biological structures of various embedded specimens. Samples were fixed, stained and embedded in resin blocks as with preparation for the transmission electron microscope (TEM). Blocks were trimmed to center the specimens in a trapezoidal face of up to 5 mm2 and their sides painted with conductive silver paint leaving the face uncovered. Blocks were sputter coated with 6-8 nm of silver, chromium or aluminum, with aluminum providing the best specimen contrast in BSE mode. Samples were examined in a field emission scanning electron microscope operated at a high emission current of 50 microA. Both the fixation protocol and microscope operating parameters were optimized to maximize the number of BSE available from the smallest probe. An accelerating voltage of 10 keV was found optimal for resolution and contrast. The technique allowed the direct visualization of embedded samples at resolutions beyond light microscopy with good contrast, without cutting sections, and avoiding grid bars obscuring areas of interest. The two dimensional images provided averaged information on the internal structures of the specimens in relation to the predicted emission depth of the BSE. The technique could be used for rapid diagnostics in pathological examinations, or for routine preselection of areas of interest within a sample face before final trimming for ultrathin sectioning for higher resolution TEM study.