Sun Mengyuan, Hu Kevin, Bewersdorf Joerg, Pollard Thomas D
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut.
Department of Biomedical Engineering, Yale University, New Haven, Connecticut.
Biophys J. 2021 Jan 5;120(1):21-34. doi: 10.1016/j.bpj.2020.11.006. Epub 2020 Nov 18.
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 three-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.
光转换荧光蛋白（PCFPs）广泛应用于超分辨率显微镜和细胞动力学研究。然而，我们对其光物理性质的了解仍然有限，这阻碍了它们的定量应用。例如，我们不知道在活细胞和固定细胞中，通过单分子定位显微镜对与PCFPs融合的蛋白质分子进行计数的最佳样品制备方法或成像条件。我们也不知道PCFPs在活细胞中的行为与固定细胞相比如何。因此，我们研究了甲醛固定在广泛的成像条件下如何影响裂殖酵母细胞中流行的绿色到红色PCFP mEos3.2的光物理性质。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随时间变化的过程中来估计光物理参数。我们发现甲醛固定可能通过使酵母细胞膜通透性增加，使得mEos3.2的荧光信号、光转换率和光漂白率对缓冲液条件敏感。在某些成像条件下，每个酵母细胞的时间积分mEos3.2信号在活细胞和在含有1 mM DTT的pH 8.5缓冲液中成像的固定细胞中相似，这表明轻度化学固定不会破坏mEos3.2分子。我们还发现405 nm的照射促使一些红色状态的mEos3.2分子进入中间暗态，该暗态可通过561 nm的光照重新转换为红色荧光态。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导。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以及光转换和光漂白率。
