How does a fluorescent protein switch in the cold ?
Structural biology methods have often benefited from their cryogenic version. X-ray crystallography and electron microscopy have been revolutionized by the possibility to flash cool biological samples, reducing radiation damage or better preserving the native state of the investigated macromolecules, respectively. With the development of super-resolution microscopy (nanoscopy), the question now arises of moving towards cryo-nanoscopy. The approach has already been demonstrated by several leading labs, notably with the goal of performing cryo-correlative studies (cryo-CLEM). There is however a big obstacle to obtain good cryo-nanoscopy images when the SMLM (single molecule localization microscopy) technique is used. For SMLM to work, the fluorophores employed to label the molecule of interest need to efficiently switch between a fluorescent on-state and a nonfluorescent off-state. However, in the case of fluorescent proteins, this switching property is typically reduced or abolished at cryogenic temperature, because switching depends on protein dynamics which is mostly arrested below the glass transition temperature.
In this work, members of the Pixel team of the Integrated Imaging of Stress Response Group (I2SR/Pixel), in collaboration with the University of Göttingen in Germany, have shown that rsEGFP2, a rapidly switching fluorescent protein at room temperature, still switches at 100K, yet based on an entirely different mechanism that likely involves radical states instead of cis trans chromophore isomerization. Most importantly, the work demonstrates that the fraction of rsEGFP2 molecules that can efficiently switch before photobleaching is significantly enhanced by using UV illumination at 355 nm instead of the classically used violet illumination at 405 nm. Thus, the study opens the door to obtaining crisper cryo-SMLM images.
Photophysical Studies at Cryogenic Temperature Reveal a Novel Photoswitching Mechanism of rsEGFP2. Angela M. R. Mantovanelli,§ Oleksandr Glushonkov,§ Virgile Adam,§ Jip Wulffelé, Daniel Thédié, Martin Byrdin, Ingo Gregor, Oleksii Nevskyi, Jörg Enderlein, and Dominique Bourgeois. Journal of the American Chemical Society 2023 ; 145(27):14636-14646. doi : 10.1021/jacs.3c01500.