Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA 15213, USA.
J Struct Biol. 2018 Jan;201(1):15-25. doi: 10.1016/j.jsb.2017.10.009. Epub 2017 Oct 25.
In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80 K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells.
在低温相关的光和电子显微镜（cryo-CLEM）中，通过荧光显微镜识别和定位冷冻感兴趣的目标，然后通过低温电子显微镜以更高的分辨率成像。在使用这些方法时，我们发现当在低温条件（80 K）下成像时，各种哺乳动物细胞会显示出强烈的点状自发荧光。自发荧光源自于多层体（MLBs）和分泌颗粒。在这里，我们描述了一种基于前者更窄的发射光谱来区分荧光蛋白标记与这些自发荧光源的方法。该方法首先在线粒体上进行测试，然后应用于检查胰岛素分泌的胰岛β细胞衍生的 INS-1E 细胞中分泌颗粒的超微结构变异性。
