Development of Fluorescent Markers for Super-Resolution Microscopy at Cryogenic Temperature

Développement de marqueurs fluorescents pour la microscopie super-résolution à température cryogénique Single-molecule localization microscopy is a powerful tool for studying biological processes at nanoscale resolution. However, in this technique, biological samples are typically fixed chemically to avoid motion blur. As the chemical fixation may produce artifacts at these high resolutions, alternative fixation strategies are of high demand. One solution is to freeze the samples at cryo-temperature, which also has other advantages as for example the possibility of performing cryo-correlative studies with cryo-EM. The main challenge of sample-freezing is the lack of fluorescent markers which can undergo efficient photo-switching at cryo-temperature. Organic dyes which are typically used in stochastic super-resolution microscopy cannot be used, because their switching is based on diffusion of STORM buffer molecules, which is hampered below the glass transition temperature. In addition, few organic fluorophores can cross the membrane of living cells, so chemical fixation is often necessary, even at cryogenic temperature. In contrast, fluorescent proteins are genetically encoded and can be expressed directly with the protein of interest and thus no fixation is necessary. Hence, they are the best candidates to be used as fluorescent markers for single-molecule localization microscopy at cryogenic temperature. However, strategies to photo-switch them more efficiently are required, as their conformational flexibility is reduced at such temperature.We performed spectroscopic and structural investigations on the fluorescent protein rsEGFP2 and also studied other proteins (rsEGFP2-V151A, rsEGFP2-V151L, mEmerald and EGFP) at room- and cryo-temperature in order to understand their switching mechanisms, and we developed an illumination strategy to enhance their recovery from non-fluorescent to fluorescent states at cryo-temperature. We observed, that for rsEGFP2, the cryo-switching mechanism is structurally and spectroscopically different from the mechanism at room temperature. In contrast to a cis-trans isomerization at room temperature, no major structural changes between the on- and off-states were observed by X-ray crystallography for switching at cryogenic temperature. The absorbance and fluorescence microspectrophotometry investigations revealed two off-states arising upon cryo-off-switching, both blue shifted as compared to the off-state which is observed after room temperature switching. With the typically used 405 nm laser only one of the off-states is recoverable to the fluorescent on-state. We observed that with a 355 nm laser both off-states are recoverable and thus achieve a substantial enhancement of total recovery of the fluorescent state. Interestingly, the switching of mEmerald and EGFP at cryo- and room-temperature appear to be very similar to the rsEGFP2 switching at cryo-temperature, which indicates that this new switching mechanism could be general to proteins of the GFP family. Using a 355 nm laser for activating fluorescent proteins like rsEGFP2, mEmerald and EGFP at cryo-temperature could open the door to more efficient effective labeling in single-molecule localization microscopy applications at cryo-temperature.