Super-resolution microscopy through single moleculelocalization at cryogenic temperature

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Super-resolution microscopy has revolutionized optical microscopy. In particular, methods based on stochastic fluorophore blinking, such as SOFI, or on single-molecule localization, such as PALM or STORM, have become widely popular. We propose to combine SOFI and PALM with cryo-fluorescence microscopy to enable imaging of shock-frozen samples with high conservation of structural detail and to open the door to correlative cryogenic optical and electron microscopy. A major bottleneck towards these goals is the lack of suitable fluorescent markers able to efficiently photo-switch at cryogenic temperatures, in particular photo-transformable fluorescent proteins (PTFPs). The core goal of our project is to design and characterize PTFPs optimized for cryo-SOFI and cryo-PALM, and to modify our existing cryo-fluorescence microscope for localizing single molecules with extreme precision and allowing cryo-CLEM studies. This research will contribute a new boost in the super-resolution field, of major interest to integrated structural and cell biology.