biostudies-literatureHirano MInformation Physics of Living MattersGrant-in-Aid for Scientific ResearchGrant-in-Aid for Scientific Research (S)the Brain Mapping by Integrated Neurotechnologies for Disease Studies from AMEDMarine Biomass Innovation ProjectGrant-in-Aid for Innovative Areas: Resonance BioJapan Science and Technology Agency Core Research for Evolutionary Science and Technology program, "Spatiotemporal dynamics of intracellular components"Japan Science and Technology Agency Core Research for Evolutionary Science and Technology program, "Spatiotemporal dynamics of intracellular components"5541https://www.ebi.ac.uk/biostudies/studies/S-EPMC10918099biostudies-literatureUnknown14(1)StayGold is a bright fluorescent protein (FP) that is over one order of magnitude more photostable than any of the currently available FPs across the full range of illumination intensities used in widefield microscopy and structured illumination microscopy, the latter of which is a widefield illumination-based technique. To compare the photostability of StayGold under other illumination modes with that of three other green-emitting FPs, namely EGFP, mClover3, and mNeonGreen, we expressed all four FPs as fusions to histone 2B in HeLa cells. Unlike the case of widefield microscopy, the photobleaching behavior of these FPs in laser scanning confocal microscopy (LSCM) is complicated. The outstanding photostability of StayGold observed in multi-beam LSCM was variably attenuated in single-beam LSCM, which produces intermittent and instantaneously strong illumination. We systematically examined the effects of different single-beam LSCM beam-scanning patterns on the photostability of the FPs in living HeLa cells. This study offers relevant guidelines for researchers who aim to achieve sustainable live cell imaging by resolving problems related to FP photostability. We also provide evidence for measurable sensitivity of the photostability of StayGold to chemical fixation.Scientific reportsStayGold photostability under different illumination modes.PMC10918099JP15dm0207001NFRFT-2020-0045219H0579521H0504119H0579415H05948JPM JCR20E2Miyawaki AAndo ROkada YFujiwara TYonemaru YHirano MShimozono SSugiyama MfalseStayGold photostability under different illumination modes.StayGold is a bright fluorescent protein (FP) that is over one order of magnitude more photostable than any of the currently available FPs across the full range of illumination intensities used in widefield microscopy and structured illumination microscopy, the latter of which is a widefield illumination-based technique. To compare the photostability of StayGold under other illumination modes with that of three other green-emitting FPs, namely EGFP, mClover3, and mNeonGreen, we expressed all four FPs as fusions to histone 2B in HeLa cells. Unlike the case of widefield microscopy, the photobleaching behavior of these FPs in laser scanning confocal microscopy (LSCM) is complicated. The outstanding photostability of StayGold observed in multi-beam LSCM was variably attenuated in single-beam LSCM, which produces intermittent and instantaneously strong illumination. We systematically examined the effects of different single-beam LSCM beam-scanning patterns on the photostability of the FPs in living HeLa cells. This study offers relevant guidelines for researchers who aim to achieve sustainable live cell imaging by resolving problems related to FP photostability. We also provide evidence for measurable sensitivity of the photostability of StayGold to chemical fixation.2024-01-01T00:00:00Z2024 Mar2025-04-04T12:34:57.908Z2025-04-04T12:34:57.908ZS-EPMC109180993844851110.1038/s41598-024-55213-3