Scanning Electron Microscopy


Preparative methods for scanning electron microscopy of chromosomes are dependent on the original source of material. Chromosomes extracted from unfixed metaphase cells via isolation buffers tend to show topography and surface morphology which may have been induced by the choice of isolation buffer itself. Furthermore, this type of preparation often precludes any chromosome identification, as many metaphases have been pooled, and also the chromosomes from these preparations are not suitable for the banding techniques regularly used in clinical cytogenetics. Our own approach has been to use the standard cytogenetic approach, starting with methanol-acetic acid fixed, air dried metaphase spreads, allowing both identification of individual chromosomes, and also the facility for various banding procedures such as G and C banding to be performed. Chromosomes are subsequently "reprepared" for SEM, using rehydration, glutaraldehye fixation, and osmium impregnation using Thiocarbohydrazide (TCH). This method produces chromosomes which can be examined at high resolution, without metallic coating, for their topography, surface morphology and chromatin organisation, and the changes produced by banding techniques which give rise to a structural alterations resulting in differential staining in the light microscope.

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