Autofluorescent proteins in single-molecule research: Applications to live cell imaging microscopy
Autofluorescent proteins in single-molecule research: Applications to live cell imaging microscopy
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DOI：
10.1016/s0006-3495(01)76209-1
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发表时间：
2001-05-01
影响因子：
3.4
通讯作者：
Schmidt, T
中科院分区：
文献类型：
--
作者：
Harms, GS;Cognet, L;Schmidt, T
分析了自体荧光蛋白的光谱和光物理特性，并与类黄酮进行了比较，以测试它们在活细胞单分子显微镜中的适用性。我们比较了1)单个自体荧光蛋白在人工和活体情况下发射的光子数，2)各种自体荧光蛋白的饱和强度，以及3)单个荧光团的最大发射光子，以明确它们在重复成像和动态分析中的用途。结果表明，在相关条件下，在毫秒积分周期内，自体荧光蛋白的光子发射率接近3000光子/毫秒(DsRed除外)，饱和强度为6~50kW/cm(2)，光漂白产率为10(-4)~10(-5)。通过定义检测率，得出结论：在体内的单分子研究中，黄色荧光蛋白突变体EYFP优于所有的荧光蛋白。这一发现随后被用来证明EYFP在生物物理研究中的适用性。通过跟踪EYFP在人造材料中的横向和旋转扩散，以及当EYFP结合到活细胞膜上时，EYFP被发现动态跟踪其锚定的实体。
The spectral and photophysical characteristics of the autofluorescent proteins were analyzed and compared to flavinoids to test their applicability for single-molecule microscopy in live cells. We compare 1) the number of photons emitted by individual autofluorescent proteins in artificial and in vivo situations, 2) the saturation intensities of the various autofluorescent proteins, and 3) the maximal emitted photons from individual fluorophores in order to specify their use for repetitive imaging and dynamical analysis. It is found that under relevant conditions and for millisecond integration periods, the autofluorescent proteins have photon emission rates of similar to 3000 photons/ms (with the exception of DsRed), saturation intensities from 6 to 50 kW/cm(2), and photobleaching yields from 10(-4) to 10(-5). Definition of a detection ratio led to the conclusion that the yellow-fluorescent protein mutant eYFP is superior compared to all the fluorescent proteins for single-molecule studies in vivo. This finding was subsequently used for demonstration of the applicability of eYFP in biophysical research. From tracking the lateral and rotational diffusion of eYFP in artificial material, and when bound to membranes of live cells, eYFP is found to dynamically track the entity to which it is anchored.