A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms
A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms
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DOI：
10.1042/bc20090096
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发表时间：
2010-02-01
影响因子：
2.7
通讯作者：
Labouesse, Michel
中科院分区：
文献类型：
--
作者：
Kolotuev, Irina;Schwab, Yannick;Labouesse, Michel
背景资料。CLEM(相关活细胞和电子显微镜)寻求通过不同的成像策略获得的数据之间的桥梁，通常在光学显微镜和电子显微镜之间。它已经成功地应用于细胞培养，尽管它在多细胞系统中的应用受到了定位感兴趣区(ROI)的困难的阻碍。我们开发了一种Clem技术，它使小型模型动物的处理变得容易，并且足够用于形态学和免疫电子显微镜标本的准备。虽然这种方法最初是针对秀丽线虫的样本开发的，但我们发现它对果蝇样本同样有效。它可以实时处理和观察任何复杂基因的单个动物，通过高压冷冻和平面包埋进行固定。我们的主要改进是开发了一种精确的测绘系统，大大简化并加快了1微米范围内ROI的回溯定位。这种方法可以在需要相关显微镜的情况下成功地使用，也可以方便地处理非相关的透射电子显微镜程序。我们的改进为通过电子显微镜处理的具有统计意义的数量的动物提供了治疗的可能性，并极大地简化了电子显微镜操作程序，使更广泛的研究人员更容易获得它们。我们相信，这项技术将有助于多细胞模型的相关研究，并将促进任何类型的瞬变显微镜标本制备的耗时过程。
Background information. CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest).Results. We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron-microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high-pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 mu m distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of non-correlative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron-microscopic protocols, making them more accessible to a wider range of researchers.Conclusions. We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time-demanding procedure of specimen preparation for any kind of TEM.