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

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... res 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 by permeabilizing the cell membrane. Under some imaging conditions we tested, the time-integrated mEos3.2 signal per cell is similar in live cells and fixed cells imaged in buffer at pH 8.5 with 1 mM DTT as a reducing agent, indicating that light chemical fixation does not destroy mEos3.2 molecules. We also discovered that 405-nm irradiation converts some mEos3.2 molecules from the green state to an intermediate state that requires 561-nm illumination for conversion to the red fluorescent state. Our findings provide a guide to compare quantitatively and optimize conditions for imaging and counting of mEos3.2-tagged molecules. 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 fluorescence proteins in cells.