Photophysical studies at cryogenic temperature reveal a novel photoswitching mechanism of rsEGFP2

Single-molecule-localization-microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples at the nanoscale and perform cryo-correlative studies. Genetically encoded fluorescent proteins (FPs) are markers of choice for cryo-SMLM, but their reduced conformational flexibility below the glass transition temperature hampers efficient photoswitching at low temperature. We investigated cryo-switching of rsEGFP2, one of the most efficient reversibly switchable fluorescent protein at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visible microspectrophotometry and X-ray crystallography revealed a completely different switching mechanism at ∼110 K. At this cryogenic temperature, on-off photoswitching involves the formation of 2 dark states with blue shifted absorption relative to that of the trans protonated chromophore populated at ambient temperature. Only one of these dark states can be switched back to the fluorescent state by 405 nm light, while both of them are sensitive to UV light at 355 nm. The rsEGFP2 photoswitching mechanism discovered in this work adds to the panoply of known switching mechanisms in fluorescent proteins. It suggests that employing 355 nm light in cryo-SMLM experiments using rsEGFP2 or possibly other FPs could improve the achievable effective labeling efficiency in this technique. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. * SMLM : single molecule localization microscopy; (d)STORM : stochastic optical reconstruction microscopy; PAINT : point accumulation in nanoscale topography; PALM : photoactivation localization microscopy; RT : room temperature; CT : cryogenic temperature; FPs : fluorescent proteins; RSFPs : reversibly switchable fluorescent proteins; PCFPs : photoconvertible fluorescent proteins; CLEM : correlative light and electron microscopy; EPR : electron paramagnetic resonance; ESRF : European Synchrotron Radiation Facility; NPC : nuclear pore complex. [1]: pending:yes