Quantitative determination of the reduction of phototoxicity and photobleaching by controlled light exposure microscopy
Quantitative determination of the reduction of phototoxicity and photobleaching by controlled light exposure microscopy
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DOI：
10.1111/j.1365-2818.2008.02009.x
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发表时间：
2008-07-01
影响因子：
2
通讯作者：
Manders, E. M. M.
中科院分区：
文献类型：
--
作者：
Hoebe, R. A.;Van der Voort, H. T. M.;Manders, E. M. M.
光毒性和光漂白是活细胞荧光显微镜的主要限制。它们是由激发单重态或三重态的荧光团引起的，这些荧光团产生单重态氧和其他活性氧物质。受控光曝光显微镜（CLEM）的原理是基于视场的非均匀照明，以减少激发的荧光团分子的数量。这种方法减少了2 - 10倍的光毒性和光漂白，而不会降低图像质量。光毒性和光漂白的减少取决于研究对象中的荧光团分布、显微镜的光学性质和CLEM电子学的设置。在这里，我们引入CLEM因子作为减少光毒性和光漂白的定量测量。最后，我们给出了一个指导方针，以优化CLEM的效果，而不影响图像质量。
Phototoxicity and photobleaching are major limitations in live-cell fluorescence microscopy. They are caused by fluorophores in an excited singlet or triplet state that generate singlet oxygen and other reactive oxygen species. The principle of controlled light exposure microscopy (CLEM) is based on non-uniform illumination of the field of view to reduce the number of excited fluorophore molecules. This approach reduces phototoxicity and photobleaching 2- to 10-fold without deteriorating image quality. Reduction of phototoxicity and photobleaching depends on the fluorophore distribution in the studied object, the optical properties of the microscope and settings of CLEM electronics. Here, we introduce the CLEM factor as a quantitative measure of reduction in phototoxicity and photobleaching. Finally, we give a guideline to optimize the effect of CLEM without compromising image quality.