Determination of quantitative distributions of heavy-metal stain in biological specimens by annular dark-field STEM.
It is shown that dark-field images collected in the scanning transmission electron microscope (STEM) at two different camera lengths yield quantitative distributions of both the heavy and light atoms in a stained biological specimen. Quantitative analysis of the paired STEM images requires knowledge of the elastic scattering cross sections, which are calculated from the NIST elastic scattering cross section database. The results reveal quantitative information about the distribution of fixative and stain within the biological matrix, and provide a basis for assessing detection limits for heavy-metal clusters used to label intracellular proteins. In sectioned cells that have been stained only with osmium tetroxide, we find an average of 1.2+/-0.1 Os atom per nm(3), corresponding to an atomic ratio of Os:C atoms of approximately 0.02, which indicates that small heavy atom clusters of Undecagold and Nanogold can be detected in lightly stained specimens.
Sousa
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Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Building 13, Room 3N17, 13 South Drive, Bethesda, MD 20892, USA.
Hohmann-Marriott
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Aronova
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Zhang
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Leapman
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D000460:Chlorophyta / Q000737:chemistry / Q000648:ultrastructure; D007150:Immunohistochemistry; D019216:Metals, Heavy / Q000032:analysis* / Q000737:chemistry; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D015203:Reproducibility of Results; D020524:Thylakoids / Q000737:chemistry / Q000648:ultrastructure
D019216:Metals, Heavy