Scanning Electron Microscopy


Electron-optical examination of reconstituted collagen fibrils fixed with tannic acid and/or glutaraldehyde and positively stained with heavy metal anions and cations reveals distinct changes in the high resolution staining patterns seen in TEM. Correlation with the known sequence data demonstrates that these changes are caused by (a) interaction of the fixative with certain charged groups in the collagen and (b) localized stain-exclusion effects following fixation. We have analysed the positive and negative staining patterns from glutaraldehyde-treated collagen in detail. In positive staining, uptake of staining ions is shown to be inhibited not only at residues known to interact with glutaraldehyde (lysyl, hydroxylysyl and probably histidyl side-chains) but also on other charged residues in the immediate neighbourhood of the glutaraldehyde--reactive residues. This 'stain exclusion' effect presumably caused by the presence of bulky polymeric complexes of glutaraldehyde molecules at cross-linking sites, accounts for the drastic changes in negative staining patterns following fixation. The effect seems to be amplified if both tannic acid and glutaraldehyde are used to fix the specimen. The bulky complexes of glutaraldehyde (and presumably tannic acid, if used) produce an altered mass distribution along the collagen fibrils before dehydration for electron microscopy. This gives rise to the observable intensity differences in the low-angle meridional X-ray diffraction patterns from moist fixed and unfixed tendons. Although collagen was used as a model system for these studies, the results should be relevant wherever glutaraldehyde and/or tannic acid are used to preserve biological tissues.

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