Allen T D, Jack E M, Harrison C J, Claugher D
Scan Electron Microsc. 1986(Pt 1):301-8.
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, glutaraldehyde 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.
用于染色体扫描电子显微镜的制备方法取决于材料的原始来源。通过分离缓冲液从未固定的中期细胞中提取的染色体往往会呈现出可能由分离缓冲液本身的选择所诱导的拓扑结构和表面形态。此外，这种类型的制备通常无法进行任何染色体鉴定，因为许多中期细胞被汇集在一起，而且这些制备物中的染色体也不适用于临床细胞遗传学中常用的显带技术。我们自己的方法是采用标准的细胞遗传学方法，从甲醇 - 乙酸固定、空气干燥的中期铺片开始，既能够识别单个染色体，也便于进行各种显带程序，如G带和C带显带。随后，使用复水、戊二醛固定以及用硫代碳酰肼（TCH）进行锇浸渍，将染色体“重新制备”用于扫描电子显微镜。这种方法产生的染色体可以在不进行金属镀膜的情况下以高分辨率检查其拓扑结构、表面形态和染色质组织，以及显带技术所产生的变化，这些变化会导致结构改变，从而在光学显微镜下产生差异染色。
