Scanning Microscopy


Thin continuous metal coatings and a scanning electron microscope-generated spot size in the range of the visualized particles, are necessary prerequisites if one hopes to extract high-resolution topographic information in the scanning electron microscope. Chemical fixation and dehydration in organic solvents at room temperature lead to severe ultrastructural artifacts which can be avoided by cryofixation and freeze-drying of the specimen. 0.9 to 2.7 nm thick homogeneous layers of chromium and germanium can be deposited onto the surface of cryofixed and freeze-dried specimens at high sub-zero temperatures by electron beam evaporation using "double-axis rotary shadowing". Theoretical calculations of the layer geometry of a double-axis rotary shadowed hemisphere and practical experiments on periodical test specimens demonstrate the usefulness of this technique. The resolution obtainable in an in-lens field emission scanning electron microscope is close to transmission electron microscope studies and image reconstructions of the same specimens. Double-axis rotary metal shadowed immunolabelled specimens allow the detection of small colloidal gold markers in the backscattered electron-image. High topographic resolution is obtained in the secondary electron-image.

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