Schaper A, Rössle M, Formanek H, Jovin T M, Wanner G
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, 37070, Germany.
J Struct Biol. 2000 Feb;129(1):17-29. doi: 10.1006/jsbi.1999.4203.
The surface structure of mitotic barley chromatin was studied by field-emission scanning electron microscopy (FESEM) and scanning force microscopy (SFM). Different stages of the cell cycle were accessible after a cell suspension was dropped onto a glass surface, chemical fixed, and critically point dried. Imaging was carried out with metal-coated specimen or uncoated specimen (only for SFM). The spatial contour of the chromatin could be resolved by SFM correlating to FESEM data. The experimentally determined volume of the residue chromatin during mitosis was within the range of 65-85 microm(3). A comparison with the theoretically calculated volume indicated a contribution of about 40% of internal cavities. Decondensation of chromosomes by proteinase K led to a drastic decrease in the chromosome volume, and a 3-D netlike architecture of the residue nucleoprotein material, similar to that in the intact chromosome, was obvious. Incubation of metaphase chromosomes in citrate buffer permitted access to different levels of chromatin packing. We imaged intact chromosomes in liquid by SFM without any intermediate drying step. A granular surface was obvious but with an appreciably lower resolution. Under similar imaging conditions proteinase K-treated chromosomes exhibited low topographic contrast but were susceptible to plastic deformations.
通过场发射扫描电子显微镜（FESEM）和扫描力显微镜（SFM）研究了有丝分裂大麦染色质的表面结构。将细胞悬液滴在玻璃表面，进行化学固定并临界点干燥后，可观察到细胞周期的不同阶段。使用金属镀膜标本或未镀膜标本（仅用于SFM）进行成像。通过与FESEM数据相关的SFM可以解析染色质的空间轮廓。有丝分裂期间实验测定的残留染色质体积在65 - 85立方微米范围内。与理论计算体积的比较表明内腔贡献约为40%。蛋白酶K处理使染色体解聚，导致染色体体积急剧减小，残留核蛋白物质呈现出类似于完整染色体的三维网状结构。在柠檬酸盐缓冲液中孵育中期染色体可观察到不同程度的染色质包装。我们通过SFM在液体中对完整染色体进行成像，无需任何中间干燥步骤。颗粒状表面明显，但分辨率明显较低。在类似的成像条件下，蛋白酶K处理的染色体显示出低地形对比度，但易发生塑性变形。
