Scanning Electron Microscopy


Although the physical dimensions of chromosomes are such that they fall well within the spatial resolving power of scanning electron microscopes, results in the past have been disappointing. This is most likely due to limitations in preparative techniques, coupled with the initial necessity to separate the chromosomes from the remainder of the metaphase cell. Two approaches have been employed, a; to use a variety of isolation buffers which provide bulk chromosome preparations, b; to use metaphase spreads prepared essentially as for light microscopy and re-processed for SEM. In the former, wide variations in chromosome surface topography and fibre organisation arise according to the choice of isolation buffer, and mixed populations preclude individual chromosome identification. In the latter the shortcomings in preparation can be considered the air drying that occurs during the making of spreads, and the initial use of methanol/acetic acid fixation. In our view however, these limitations in preparation are more than compensated by the ability to identify individual chromosomes, resolve chromatin fibre organisation, and compare the structural changes produced by a variety of banding techniques. Using this technique we have established a structural basis for the differential staining patterns that result from G and C banding treatments.

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