Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching
Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching
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DOI：
10.1111/j.1365-2818.2007.01794.x
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发表时间：
2007-08-01
影响因子：
2
通讯作者：
Nicastro, Daniela
中科院分区：
文献类型：
--
作者：
Schwartz, Cindi L.;Sarbash, Vasily I.;Nicastro, Daniela
荧光显微镜（LM）在研究细胞组织方面具有许多优势。标本制备简单且相对便宜，使用适当的标签使科学家能够可视化感兴趣的特定蛋白质。然而，LM的分辨率有限，因此当人们对超微结构感兴趣时，必须转向电子显微镜（EM），即使这种方法本身存在问题。细胞EM的最大困难是其在定位感兴趣的大分子同时保持良好的结构保存方面的有限效用。我们已经建立了一个冷冻光显微镜阶段，使我们能够生成为冷冻EM准备的玻璃体样品的LM图像。相关的LM和EM允许人们通过使用荧光蛋白或活体染料作为玻璃化样品内的标记物来找到特别感兴趣的区域。一旦定位，样品可以被放置在EM中，以更高的分辨率进行进一步研究。低温LM阶段的另一个好处是，光漂白在低温（-140摄氏度）下比在室温下慢。
Fluorescence light microscopy (LM) has many advantages for the study of cell organization. Specimen preparation is easy and relatively inexpensive, and the use of appropriate tags gives scientists the ability to visualize specific proteins of interest. LM is, however, limited in resolution, so when one is interested in ultrastructure, one must turn to electron microscopy (EM), even though this method presents problems of its own. The biggest difficulty with cellular EM is its limited utility in localizing macromolecules of interest while retaining good structural preservation. We have built a cryo-light microscope stage that allows us to generate LM images of vitreous samples prepared for cryo-EM. Correlative LM and EM allows one to find areas of particular interest by using fluorescent proteins or vital dyes as markers within vitrified samples. Once located, the sample can be placed in the EM for further study at higher resolution. An additional benefit of the cryo-LM stage is that photobleaching is slower at cryogenic temperatures (- 140 degrees C) than at room temperature.