Photoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy and studies of cellular dynamics. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, we do not know the optimal sample preparation methods or imaging conditions to count protein molecules fused to PCFPs by single-molecule localization microscopy in live and fixed cells. We also do not know how the behavior of PCFPs in live cells compares with fixed cells. Therefore, we investigated how formaldehyde fixation influences the photophysical properties of the popular green-to-red PCFP mEos3.2 in fission yeast cells under a wide range of imaging conditions. We estimated photophysical parameters by fitting a 3-state model of photoconversion and photobleaching to the time course of fluorescence signal per yeast cell expressing mEos3.2. We discovered that formaldehyde fixation makes the fluorescence signal, photoconversion rate and photobleaching rate of mEos3.2 sensitive to the buffer conditions likely by permeabilizing the yeast cell membrane. Under some imaging conditions, the time-integrated mEos3.2 signal per yeast cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that 405-nm irradiation drove some red-state mEos3.2 molecules to enter an intermediate dark state, which can be converted back to the red fluorescent state by 561-nm illumination. Our findings provide a guide to quantitatively compare conditions for imaging mEos3.2-tagged molecules in yeast cells. Our imaging assay and mathematical model are easy to implement and provide a simple quantitative approach to measure the time-integrated signal and the photoconversion and photobleaching rates of fluorescent proteins in cells.

中文翻译：

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样品制备和成像条件影响裂殖酵母细胞中的 mEos3.2 光物理学

光转换荧光蛋白 (PCFP) 广泛用于超分辨率显微镜和细胞动力学研究。然而，我们对其光物理学的了解仍然有限，阻碍了它们的定量应用。例如，我们不知道通过单分子定位显微镜在活细胞和固定细胞中计算融合到 PCFP 的蛋白质分子的最佳样品制备方法或成像条件。我们也不知道活细胞中 PCFP 的行为与固定细胞相比如何。因此，我们研究了在各种成像条件下，甲醛固定如何影响裂殖酵母细胞中流行的绿色到红色 PCFP mEos3.2 的光物理特性。我们通过将光转换和光漂白的三态模型拟合到每个表达 mEos3.2 的酵母细胞的荧光信号的时间过程来估计光物理参数。我们发现甲醛固定可能通过透化酵母细胞膜使 mEos3.2 的荧光信号、光转化率和光漂白率对缓冲条件敏感。在某些成像条件下，每个酵母细胞的时间积分 mEos3.2 信号在活细胞和固定细胞中在 pH 8.5 和 1 mM DTT 的缓冲液中成像相似，表明光化学固定不会破坏 mEos3.2 分子。我们还发现 405 nm 照射会驱使一些红态 mEos3.2 分子进入中间暗态，可以通过 561 nm 照射将其转换回红色荧光态。我们的研究结果为定量比较酵母细胞中 mEos3.2 标记分子的成像条件提供了指南。我们的成像分析和数学模型易于实施，并提供了一种简单的定量方法来测量细胞中荧光蛋白的时间积分信号和光转化率和光漂白率。