Exploring Transient States of PAmKate to Enable Improved Cryogenic Single-Molecule Imaging.

Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nanoscale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) that photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remain a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find that a transient state forms with a lifetime of ∼2 ms under cryogenic conditions, which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for the study of other FPs at cryogenic temperatures.