Division of Gynecologic Oncology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305.
Institut Curie, PSL Research University, Cellular and Chemical Biology, INSERM U 1143, CNRS UMR 3666, 26 rue d'Ulm, 75248 Paris Cedex 05, France.
Cytometry A. 2018 Apr;93(4):411-419. doi: 10.1002/cyto.a.23319. Epub 2017 Dec 29.
Photoactivatable fluorescent proteins (PA-FPs) have been widely used to assess the dynamics of cell biological processes. In addition, PA-FPs enabled single-molecule based super-resolution imaging (photoactivated localization microscopy) and thereby provided unprecedented structural insight. For the lack of tools, however, the fraction of PA-FPs that is, actually being switched on to fluoresce, that is, the photoactivation efficiency, has been difficult to assess. Uncertainty about photoactivation efficiency has hampered an understanding of the absolute amount of PA-FPs, that is, being examined. Here, we present internal rulers to assess photoactivation efficiencies of photoactivatable proteins. These internal rulers comprise a PA-FP that is genetically directly coupled to a spectrally distinct always-on fluorescent protein. Thus, these fluorescent proteins will be expressed in the bacterial and mammalian cell in a one-to-one ratio. With these tools, we describe photoactivation efficiencies of PA-GFP and PA-Cherry in intensity-based ratiometric ensemble studies and on the single-molecule level. In ratiometric ensemble studies, we show that photoactivation efficiency depends on how the PA-FPs are exposed to 405 nm light. Using a laser-scanning microscope, hundreds of iterative low-level exposures are up to four times more efficient than a short high-power exposure. Using wide-field illumination, photoactivation was similarly efficient and instantaneous. These findings suggest that the repetitive or stochastic exposure to photons of 405 nm light results in more efficient photoactivation than a continuous flow of photons. Because of the differential photoactivation efficiency, it is crucial to assess photoactivation efficiency for any given experimental set-up. The tools we provide can be applied to any genetically encoded photoactivatable protein. Determination of photoactivation efficiency is essential for an understanding of absolute molecule numbers in ensemble studies and, most importantly, quantitative superresolution imaging. © 2017 International Society for Advancement of Cytometry.
光激活荧光蛋白（PA-FPs）已被广泛用于评估细胞生物学过程的动态。此外，PA-FPs 使基于单分子的超分辨率成像（光激活定位显微镜）成为可能，从而提供了前所未有的结构洞察力。然而，由于缺乏工具，实际被激活荧光的 PA-FPs 分数，即光激活效率，难以评估。光激活效率的不确定性阻碍了对被检查的 PA-FPs 的绝对数量的理解。在这里，我们提出了内部标尺来评估光激活蛋白的光激活效率。这些内部标尺包括一个与光谱上明显的始终处于激活状态的荧光蛋白直接遗传偶联的 PA-FP。因此，这些荧光蛋白将在细菌和哺乳动物细胞中以一比一的比例表达。有了这些工具，我们描述了 PA-GFP 和 PA-Cherry 在基于强度的比率整体研究和单分子水平上的光激活效率。在比率整体研究中，我们表明光激活效率取决于 PA-FPs 如何暴露于 405nm 光。使用激光扫描显微镜，数百次迭代的低水平曝光比短时间的高功率曝光效率提高了四倍。使用宽场照明，光激活效率相似且瞬时。这些发现表明，与连续的光子流相比，重复或随机暴露于 405nm 光的光子会导致更有效的光激活。由于光激活效率的差异，对于任何给定的实验设置，评估光激活效率至关重要。我们提供的工具可应用于任何基因编码的光激活蛋白。确定光激活效率对于理解整体研究中的绝对分子数量以及最重要的是定量超分辨率成像至关重要。 © 2017 国际细胞分析协会。
