biostudies-literatureSun MNIBIB NIH HHSNational Institutes of HealthWellcome TrustNIGMS NIH HHS21-34https://www.ebi.ac.uk/biostudies/studies/S-EPMC7820738biostudies-literatureUnknown120(1)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 photocoBiophysical journalSample Preparation and Imaging Conditions Affect mEos3.2 Photophysics in Fission Yeast Cells.PMC7820738203285/B/16/ZR01 GM026132T32 EB019941R01 GM118486Sun MPollard TDBewersdorf JHu KfalseSample Preparation and Imaging Conditions Affect mEos3.2 Photophysics in Fission Yeast Cells.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 photoco2021-01-01T00:00:00Z2021 Jan2025-04-20T03:42:48.283Z2025-04-20T03:42:48.283ZS-EPMC78207383321738110.1016/j.bpj.2020.11.006