Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging
Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging
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DOI：
10.1038/nbt1278
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发表时间：
2007-02-01
影响因子：
46.9
通讯作者：
Manders, E. M. M.
中科院分区：
文献类型：
--
作者：
Hoebe, R. A.;Van Oven, C. H.;Manders, E. M. M.
活细胞的荧光显微镜能够可视化细胞内分子的动态和相互作用。然而，荧光活细胞成像受到激发光引起的光漂白和光毒性的限制。在这里，我们描述受控光曝光显微镜(CLEM)，这是一种简单的成像方法，可以将光漂白和光毒性减少两到十倍，这取决于荧光团在对象中的分布。通过空间控制曝光时间，Clem在不影响图像质量的情况下减少了激发光剂量。我们发现，在表达微管相关GFP-MAP4的烟草植物细胞中，Clem将光漂白减少了七倍，在表达染色质相关的H2B-GFP的HeLa细胞中，Clem将活性氧的产生减少了八倍，并将细胞存活延长了六倍。此外，Clem将荧光强度的动态范围增加了至少两倍。
Fluorescence microscopy of living cells enables visualization of the dynamics and interactions of intracellular molecules. However, fluorescence live-cell imaging is limited by photobleaching and phototoxicity induced by the excitation light. Here we describe controlled light-exposure microscopy ( CLEM), a simple imaging approach that reduces photobleaching and phototoxicity two- to tenfold, depending on the fluorophore distribution in the object. By spatially controlling the light-exposure time, CLEM reduces the excitation-light dose without compromising image quality. We show that CLEM reduces photobleaching sevenfold in tobacco plant cells expressing microtubule-associated GFP-MAP4 and reduces production of reactive oxygen species eightfold and prolongs cell survival sixfold in HeLa cells expressing chromatin-associated H2B-GFP. In addition, CLEM increases the dynamic range of the fluorescence intensity at least twofold.