Sanchez-Sweatman O H, de Harven E P, Dubé I D
Department of Pathology, University of Toronto, Canada.
Scanning Microsc. 1993 Mar;7(1):97-104; discussion 105-6.
Methods for scanning electron microscopy (SEM) of chromosomes have been developed in the last two decades. Technical limitations in the study of human chromosomes, however, have hindered the routine use of SEM in clinical and experimental human cytogenetics. We compared different methodologies, including metal impregnation, air drying and specimen coating. SEM preparation of human chromosomes in which osmium impregnation is mediated by tannic acid, yielded more reproducible results when compared with osmium impregnation protocols previously described. The level of osmium impregnation was systematically evaluated by imaging chromosomes in the backscattering mode. Critical point drying and a light gold-palladium coating were essential for appropriate secondary electron imaging of chromosomes. With this method, and in a preliminary quantitative analysis, we show that our SEM technique is more sensitive than light microscopy for the detection of aphidicolin-induced fragile sites. This technical approach is useful for chromosomal studies requiring resolution higher than that obtained by light microscopy. Also, it allows the use of clinical and archival chromosomal samples prepared by routine cytogenetic techniques.
在过去二十年中已开发出用于染色体扫描电子显微镜（SEM）的方法。然而，人类染色体研究中的技术限制阻碍了SEM在临床和实验人类细胞遗传学中的常规应用。我们比较了不同的方法，包括金属浸染、空气干燥和标本镀膜。与先前描述的锇浸染方案相比，用单宁酸介导锇浸染的人类染色体SEM制备产生了更可重复的结果。通过在背散射模式下对染色体成像，系统地评估了锇浸染的水平。临界点干燥和浅金钯镀膜对于染色体适当的二次电子成像至关重要。通过这种方法，并在初步定量分析中，我们表明我们的SEM技术在检测阿非科林诱导的脆性位点方面比光学显微镜更敏感。这种技术方法对于需要高于光学显微镜分辨率的染色体研究很有用。此外，它允许使用通过常规细胞遗传学技术制备的临床和存档染色体样本。
