A Red Fluorescent Protein for Cryogenic Single-Molecule Superresolution Imaging

Superresolution imaging techniques routinely resolve fluorescently labelled structures in cells with resolution more than an order of magnitude beyond the diffraction limit. The molecular specificity and spatial resolution of these techniques make them a powerful tool for the life sciences. In single-molecule superresolution imaging, the precision with which a single molecule can be localized depends on the number of photons collected before photobleaching. It has long been known that photobleaching is significantly reduced at cryogenic temperatures; thus, cryogenic fluorescence imaging can provide a route to improved superresolution imaging. However, performing superlocalization measurements based on stochastic activation of single fluorophores (e.g. PALM, STORM) requires the fluorophores to exhibit the necessary photoswitching properties at cryogenic temperatures. Restricted conformational mobility in a frozen solvent prevents many photoactivatable small molecule fluorophores and fluorescent proteins from undergoing the necessary isomerizations or other rearrangements required for photoactivation or blinking. In this work, we have identified a red fluorescent protein which exhibits photoactivation at cryogenic temperatures. Using a custom fluorescence microscope capable of maintaining a sample at cryogenic temperatures during imaging, we have characterized the photoswitching behaviour and obtained superresolution images of previously studied proteins in the model organism Caulobacter crescentus.