Factors affecting preparation of chromosomes for scanning electron microscopy using osmium impregnation

Osmium impregnation techniques have become useful for imparting conductivity to tissue specimens for SEM, thereby avoiding coating with gold or other metals. Such techniques have been developed to produce aesthetically pleasing images of mammalian (particularly human) chromosomes prepared by standard cytogenetical methods which use methanol-acetic acid fixation. The present study was designed: (1) to examine changes in the appearance of chromosomes as a result of preparation by osmium impregnation techniques; (2) to assess the function and importance of the various stages of chromosome preparation; and (3) to identify the chemical groups responsible for osmium binding. Methanol-acetic acid fixed chromosomes are known to have lost many proteins during fixation, and appear to be flattened down on the substrate. Osmium impregnation swells these flattened chromosomes to a variable extent, but the result is inevitably an artefact, albeit a useful one, and not a true representation of the chromosome in vivo. The size of chromatin fibres, for example, is the consequence of the degree of protein extraction during fixation, the loss of material during pre-treatments (e.g. trypsin), and the amount of osmium uptake during impregnation. Trypsin pre-treatment removes a surface coating of protein from the chromosomes as well as exposing chemical groups which can react with osmium. The principal reactive site appears to be amino groups, which bind glutaraldehyde, which in turn binds thiocarbohydrazide, to which the osmium becomes attached. Pre-treatments other than trypsin can be used to extract chromosomal material and to reveal different aspects of chromosome structure.