Sample Preparation and Imaging Conditions Affect mEos3.2 Photophysics in Fission Yeast Cells

Abstract

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 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 some red-state mEos3.2 molecules entered an intermediate dark state that is converted back to the red fluorescent state by 561-nm illumination. Our findings provide a guide to compare quantitatively conditions for imaging and counting of 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. STATEMENT OF SIGNIFICANCEMaking quantitative measurements with single-molecule localization microscopy (SMLM) has been impeded by limited understanding of the photophysics of the fluorophores, which is very sensitive to the sample preparation and imaging conditions. We characterized the photophysics of the green-to-red photoconvertible fluorescent protein mEos3.2, which is widely used in SMLM. We combined quantitative fluorescence microscopy and mathematical modeling to measure the fluorescence signal and rate constants for photoconversion and photobleaching of mEos3.2 in live and fixed cells under a wide range of illumination intensities. Our findings

Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties

“…[ 38 ] This discrepancy may possibly be explained by the presence of strong additional illumination at 561 nm in optical microscopy experiments, resulting in enhanced photoconversion efficiencies. [ 39 ] We thus cannot exclude that photoconversion of mEos4b is based on a more complex scenario than the simple model proposed in Figure 8B , involving additional photo‐induced transformations and states. Future NMR experiments in the presence of both illumination lasers will allow to address this issue.…”

mEos4b Photoconversion Efficiency Depends on Laser Illumination Conditions Used in PALM

“…To test this, we monitored the progression of ensemble mEos4b red fluorescence under 1, 10, and 100 W/ cm 2 405 nm illumination and weak exposure to 561 nm light (8 W/cm 2 ) (Figure 3A). Sun et al 7 reported that the ensemble red fluorescence intensity of mEos3.2 under constant 405 and 561 nm illumination follows a three-state model: green → red → bleached. Such a model implies that the level of the maximum recorded red fluorescence increases with the applied 405 nm light intensity as bleaching by the 561 nm laser lowers the peak height at low 405 nm intensities.…”

“…However, a disadvantage of mEos-based PCFPs is their highly complex photophysical behavior (Figure S1). − …”

mEos4b photoconversion efficiency depends on laser illumination conditions used in PALM

“…3 However, a disadvantage of mEos-based PCFPs is their highly complex photophysical behavior (Supplementary Figure S1). [4][5][6][7] One property essential to the success of PALM experiments using PCFPs is the so-called photoconversion efficiency (PCE), 8,9 sometimes also referred to as signaling efficiency. 10 Typically, upon prolonged violet light illumination, green-to-red photoconversion remains incomplete, meaning that from a pool of PCFPs, only a limited fraction can ever be imaged in the red channel of the microscope.…”