Photo-switching Quantum Yield of Reversibly Switchable Fluorescent Proteins depends on Wavelength and Temperature

Reversibly switchable fluorescent proteins (RSFPs) enable super-resolved bio-imaging by leveraging the photo-switching between their On-and Off-forms. The photoswitching quantum yield (φ) is the key parameter that characterizes the switching efficiency of RSFPs, yet reported values vary widely. Although, the quantum yield of molecular organic photo-switches is known to depend on both temperature and excitation wavelength, these dependencies have often been overlooked in studies of RSFPs. To study the modulation of φ, the wild type rsEGFP2 as well as two of its variants were selected. The wild type rsEGFP2 is a representative RSFP that has been reported multiple times in the literature of nanoscopy, and exibits up to four-fold variation in the reported values of φ. We measured the dependence of values on temperature (from 10 to 35°C) and on irradiation wavelength (408 and 480 nm) across variants, revealing evidence of distinct cis-trans excited state isomerization pathways. These results suggest that temperature-and wavelength-dependence are general properties of RSFPs and may offer new insights into their photo-switching mechanisms, enabling the development of novel temperature-and color-modulated imaging strategies.