The Role of Probe Photophysics in Localization-Based Superresolution Microscopy
The Role of Probe Photophysics in Localization-Based Superresolution Microscopy
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DOI：
10.1016/j.bpj.2017.08.054
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发表时间：
2017-11-07
影响因子：
3.4
通讯作者：
Hess, Samuel T.
中科院分区：
文献类型：
--
作者：
Pennacchietti, Francesca;Gould, Travis J.;Hess, Samuel T.
荧光蛋白广泛用于生物成像应用；光激活和光转换荧光蛋白（PAFPs）广泛应用于荧光光激活定位显微镜和光激活定位显微镜等超分辨率定位显微镜方法中。然而，它们的最佳使用不仅取决于它们的整体荧光特性，还取决于它们在单分子水平上的光物理特性。我们利用荧光相关光谱和互相关光谱对定位显微镜中使用的著名PAFP Dendra2的扩散、光漂白、荧光间歇和光转换动力学进行了量化。在无活性（绿色荧光）和活性（橙色荧光）形式下，可以观察到Dendra2的许多暗态；与其他pafp观察到的结果一样，相互转化率与光和ph有关。暗态限制了每个分子的检测计数率，这是定位显微镜的一个关键参数。然后，据我们所知，我们开发了一种新的数学估计，用于定位显微镜的分辨率，作为测量探针的光物理参数的函数，如光漂白量子产率，每个分子的计数率和饱和度。该模型用于预测分辨率对采集参数（如照明强度和时间帧）的依赖性，展示了针对各种分辨率测量的给定探针的最佳采集参数集。然后比较了Dendra2和其他广泛使用的探针的最佳分辨率，包括Alexa染料和量子点。这项工作建立了一个框架，用于确定使用定位显微镜成像特定荧光团的最佳可能分辨率，并建议用于超分辨率定位显微镜的探针的开发必须考虑每个分子的计数率，饱和强度，光漂白产量，以及至关重要的，亮/暗状态转换的管理，以优化图像分辨率。
Fluorescent proteins are used extensively for biological imaging applications; photoactivatable and photoconvertible fluorescent proteins (PAFPs) are used widely in superresolution localization microscopy methods such as fluorescence photoactivation localization microscopy and photoactivated localization microscopy. However, their optimal use depends on knowledge of not only their bulk fluorescence properties, but also their photophysical properties at the single molecule level. We have used fluorescence correlation spectroscopy and cross-correlation spectroscopy to quantify the diffusion, photobleaching, fluorescence intermittency, and photoconversion dynamics of Dendra2, a well-known PAFP used in localization microscopy. Numerous dark states of Dendra2 are observed both in inactive (green fluorescent) and active (orange fluorescent) forms; the interconversion rates are both light-and pH-dependent, as observed for other PAFPs. The dark states limit the detected count rate per molecule, which is a crucial parameter for localization microscopy. We then developed, to our knowledge, a new mathematical estimate for the resolution in localization microscopy as a function of the measured photophysical parameters of the probe such as photobleaching quantum yield, count rate per molecule, and intensity of saturation. The model was used to predict the dependence of resolution on acquisition parameters such as illumination intensity and timeper frame, demonstrating an optimal set of acquisition parameters for a given probe for a variety of measures of resolution. The best possible resolution was then compared for Dendra2 and other widely used probes, including Alexa dyes and quantum dots. This work establishes a framework for determination of the best possible resolution using a localization microscope to image a particular fluorophore, and suggests that development of probes for use in superresolution localization microscopy must consider the count rate per molecule, the saturation intensity, the photobleaching yield, and, crucially, management of bright/dark state transitions, to optimize image resolution.