Celebrating 50 years of fluorescence correlation spectroscopy (FCS): Advancing live-cell massively parallel FCS studies with photostable GFPs, mStayGold and StayGold/E138D

More than 50 years after its inception, fluorescence correlation spectroscopy (FCS) remains a cornerstone technique for quantitative characterization of the cellular dynamics of molecules and their concentration and interactions in live cells. The enhanced green fluorescent protein (eGFP) has long been a preferred tag in live-cell FCS, valued for its brightness, photostability and lack of posttranslational modifications. However, low eGFP photostability limits measurement durations, posing challenges for studying dynamic cellular processes necessitating longer measurement time. Recent advancements in fluorescent protein engineering have yielded mStayGold and StayGold/E138D, two highly photostable monomeric GFP variants. In this study, we evaluate their performance in live cells and utility for FCS by quantifying glucocorticoid receptor (GR) homodimerization and nuclear import/export dynamics in live cells. Our study shows that both mStayGold and StayGold/E138D exhibit twice the brightness of eGFP, significantly enhancing the signal-to-noise ratio (SNR). Using massively parallel FCS (mpFCS) and two-foci cross-correlation to characterize the direction of GR nucleocytoplasmic transport along the nuclear envelope, we also confirm that these proteins show significantly improved photostability over eGFP.