Single-molecule localization microscopy (SMLM) improves the resolution of fluorescence microscopy normally limited by diffraction. One challenge is to work at cryogenic temperature to preserve the native structure of the investigated samples, like in cryo electron microscopy. The “magic” of SMLM lies in the properties of the employed fluorophores used to label the biological target. Those fluorophores are able to efficiently switch between a fluorescent on-state and a nonfluorescent off-state. However photoswitching is killed at very low temperature.
In response, researchers at IRIG, in collaboration with the University of Göttingen in Germany, investigated the cryo-switching properties of rsEGFP2, a fluorescent protein, combining X-ray crystallography with optical spectroscopy and SMLM using a dedicated microscope operating at cryogenic temperature. They found that rsEGFP2 still switches at - 170 °C, based on a photophysical mechanism different from that observed at room temperature. Whereas at room temperature switching is based on cis-trans isomerization of the chromophore, a large conformational change, the data suggest that cryo-switching involves the formation of a so-called “radical states” without any substantial conformational change (see figure).
Moreover, the researchers found that the fraction of rsEGFP2 molecules that can efficiently cryo-switch before photo destruction (photobleaching) is enhanced by about 30% using weak UV illumination at 355 nm instead of 405 nm as classically used for SMLM at room temperature. Thus 355 nm light substantially improves the effective labeling density in cryo-SMLM.
The study opens the door to obtaining crisper cryo-nanoscopy images. Applying the optimized UV illumination to real biological samples is now the goal, notably in the context of cryo-correlative studies between SMLM and Focused Ion Beam for Scanning Electron Microscopy and electron tomography, a top challenge for today’s integrated structural cell biology .
Figure: Conformational changes of the rsEGFP2 chromophore upon switching at room temperature (left) or at cryogenic temperature (right). The fluorescence on-state is shown in green and the nonfluorescent off-state in grey. An artistic view of a cryo-SMLM microscope is shown in the background. © Virgile Adam / IBS