Photophysical Studies at Cryogenic Temperature Reveal a Novel Photoswitching Mechanism of rsEGFP2

Single-moleculelocalization microscopy (SMLM) at cryogenic temperatureopens new avenues to investigate intact biological samples at thenanoscale and perform cryo-correlative studies. Genetically encodedfluorescent proteins (FPs) are markers of choice for cryo-SMLM, buttheir reduced conformational flexibility below the glass-transitiontemperature hampers efficient cryo-photoswitching. We investigatedcryo-switching of rsEGFP2, one of the most efficient reversibly switchablefluorescent proteins at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visiblemicrospectrophotometry and X-ray crystallography revealed a completelydifferent switching mechanism at & SIM;110 K. At this cryogenictemperature, on-off photoswitching involves the formation oftwo off-states in cis conformation with blue-shiftedabsorption relative to that of the trans protonatedchromophore populated at ambient temperature. Only one of these off-statescan be switched back to the fluorescent on-state by 405 nm light,while both of them are sensitive to UV light at 355 nm. Superior recoveryto the fluorescent on-state by 355 nm light was confirmed at the single-moleculelevel. This suggests, as also shown by simulations, that employing355 nm light in cryo-SMLM experiments using rsEGFP2 and possibly otherFPs could improve the effective labeling efficiency achievable withthis technique. The rsEGFP2 photoswitching mechanism discovered inthis work adds to the panoply of known switching mechanisms in fluorescentproteins.