Heavy metal compounds are adsorbed onto biological specimen in order to enhance the contrast as well as to preserve the structural features of the specimen against electron beam-induced radiation damage. In particular, in combination with computational image processing, negative staining is widely used for structural analysis of protein complexes to moderate resolutions. Image analysis of negatively stained biological specimen is known to suffer from limited achievable resolution due to dehydration and large grain size of staining molecules although the extent of such effect remains somewhat dubious. Stain molecules exist as grains under electron beam. However, clear observation of the crystalline nature of the grains and their association with biological specimen has not been thoroughly demonstrated. In this study, we attempted high-resolution TEM (HRTEM) using high voltage electron microscopy and electron crystallography analysis for the detailed characterization of negatively stained biological specimen, focusing on physical state and chemical composition of the stain molecules. The electron crystallography analysis allowed for the identification of the crystal constituents of widely used stains, hence revealing the chemical nature and the morphology of the stain molecules at specimen level. This study re-evaluated generally accepted notions on negative staining, and may help correctly interpreting the structural analysis of stained biological specimen.
Keywords: Electron crystallography; High voltage electron microscopy; Nano-crystal; Negative staining; Transmission electron microscopy; Uranium dioxide.
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