Merdes A, Stelzer E H, De Mey J
European Molecular Biology Laboratory, Heidelberg, Germany.
J Electron Microsc Tech. 1991 May;18(1):61-73. doi: 10.1002/jemt.1060180110.
Fluorescence microscopy techniques have become important tools in mitosis research. The well-known disadvantages of fluorescence microscopy, rapid bleaching, phototoxicity and out-of-focus contributions blurring the in-focus image are obstacles which still need to be overcome. Confocal fluorescence microscopy has the potential to improve our capabilities of analyzing cells, because of its excellent depth-discrimination and image processing power. We have been using a confocal fluorescence microscope for the study of the mechanism of poleward chromosome movement, and report here 1) a cell preparation technique, which allows labeling of fixation sensitive spindle antigens with acceptable microtubule preservation; 2) the use of image processing methods to represent the spatial distribution of various labeled elements in pseudocolour; 3) a novel immunoelectron microscopic labeling method for microtubules, which allows the visualization of their distribution in semithin sections at low magnification; and 4) a first attempt to study microtubule dynamics with a confocal fluorescence microscope in living cells, microinjected with rhodamine labeled tubulin. Our experience indicates that confocal fluorescence microscopy provides real advantages for the study of spatial colocalization of antigens in the mitotic spindle. It does not, however, overcome the basic limits of resolution of the light microscope. Therefore, it has been necessary to use an electron microscopic method. Our preliminary results with living cells show that it is possible to visualize the entire microtubule network in stereo, but that the sensitivity of the instrument is still too low to perform dynamic time studies. It will be worthwhile to further develop this new type of optical instrumentation and explore its usefulness on both fixed and living cells.
荧光显微镜技术已成为有丝分裂研究中的重要工具。荧光显微镜存在一些众所周知的缺点，如快速漂白、光毒性以及离焦信号干扰聚焦图像，这些仍是需要克服的障碍。共聚焦荧光显微镜因其出色的深度分辨能力和图像处理能力，有潜力提升我们分析细胞的能力。我们一直在使用共聚焦荧光显微镜研究染色体向极运动的机制，在此报告：1）一种细胞制备技术，该技术能在保留可接受的微管状态下标记对固定敏感的纺锤体抗原；2）使用图像处理方法以伪彩色呈现各种标记元素的空间分布；3）一种针对微管的新型免疫电子显微镜标记方法，该方法能在低倍放大的半薄切片中观察微管的分布；4）首次尝试用共聚焦荧光显微镜研究注射了罗丹明标记微管蛋白的活细胞中的微管动力学。我们的经验表明，共聚焦荧光显微镜在研究有丝分裂纺锤体中抗原的空间共定位方面具有实际优势。然而，它并未克服光学显微镜分辨率的基本限制。因此，有必要使用电子显微镜方法。我们对活细胞的初步结果表明，有可能以立体方式观察整个微管网络，但仪器的灵敏度仍过低，无法进行动态时间研究。进一步开发这种新型光学仪器并探索其在固定细胞和活细胞上的实用性将是值得的。
