Engineering of a bacteriophytochrome-derived reversibly switchable fluorescent protein for the application in super-resolution microscopy in the near-infrared window

The developments of fluorescence microscopy techniques has advanced our understanding of biological systems on a cellular level. In recent years, several super-resolution microscopy techniques overcame the diffraction barrier thereby allowing unprecedented insights in the subcellular structures down to the molecular level. Reversible saturable optical linear fluorescence transition (RESOLFT) microscopy, as one of these techniques, allows for the formation of super-resolved images by utilizing low light intensities in the kW/cm2 range, which makes it a suitable tool for live cell super-resolution imaging. The RESOLFT concept relies on-switching fluorophores between a fluorescent on- and non-fluorescent off-state. To date, most RESOLFT applications depend on reversibly switchable fluorescent proteins (RSFP), which are excited and switched by light in the visible region of the electromagnetic spectrum. In order to transfer RESOLFT to the near-infrared (NIR) region of the electromagnetic spectrum, which exhibits reduced scattering and phototoxicity, a new class of RSFPs is required. Bacteriophytochromes as NIR-photoreceptors in bacteria act as suitable templates for engineering a NIR-RSFP. In this work, the monomeric bacteriophytochrome-derived PhotostablE Nir rEversibly switchable fLuOrescent ProteEin (PENELOPE) was engineered using site-directed and random mutagenesis. PENELOPE exhibits a strong photostability, fast off-switching and allows RESOLFT imaging in the NIR window for the first time. NIR-RESOLFT microscopy was performed by using a single wavelength. This was enabled by harnessing the remarkable ability of PENELOPE to thermally relax to the on-state within milliseconds after off-switching. This allowed the substitution of the conventional on-switching step with a thermal relaxation step, which additionally reduced the applied light doses during imaging. Furthermore, RESOLFT microscopy was successfully performed in fixed mammalian cells using a genetically encoded RSFP for the first time. The implementation of a time-saving pixel hop scan approach in conjunction with a novel proposed switching state model for PENELOPE, ushers PENELOPE into low light dose NIR super-resolution microscopy.2023-06-0