2025
PNAS

The near-infrared bacteriophytochrome-derived fluorescent protein PENELOPE enables RESOLFT superresolution microscopy

Authors:

Daniel Stumpf, Nickels Jensen, Cédric Mittelheisser, Jan Keller-Findeisen, Alexey I. Chizhik, Maria Kamper, Timo Diekmann, Florian Habenstein, Isabelle Jansen, Jörg Enderlein, Michel Sliwa, Kaushik Inamdar, Stefan W. Hell, and Stefan Jakobs

Keywords:

superresolution microscopy; RESOLFT; fluorescent proteins; live-cell imaging; RSFP; PENELOPE; near-infrared

Abstract:

REversible Saturable Optical Linear Fluorescence Transitions (RESOLFT) superresolution microscopy fundamentally overcomes the diffraction barrier in far-field fluorescence microscopy. It relies on reversibly switchable fluorescent proteins (RSFPs) that allow repeated light-induced transitions between fluorescent on- and nonfluorescent off-states. Because these transitions are induced by low-light intensities, RESOLFT superresolution microscopy is particularly suitable for live-cell imaging. So far, RESOLFT imaging has only been performed in the visible range of the electromagnetic spectrum. To expand the RESOLFT concept into the near-infrared (NIR) region, which is characterized by reduced autofluorescence, lower scattering and decreased phototoxicity, we developed the photostable NIR reversibly switchable fluorescent protein (PENELOPE), which is the first RSFP applicable in the NIR window. PENELOPE was generated by mutagenesis of the chromophore-binding domain of the Deinococcus radiodurans bacteriophytochrome. This NIR-RSFP exhibits high photostability and high ensemble switching contrast at low-light intensities. It also undergoes an unusually fast thermal fluorescence recovery from the dark state into an on-state. This was exploited for low-light intensity RESOLFT imaging with only a single wavelength, as the same light wavelength (660 nm) is used for off-switching and fluorescence readout, while the on-switching occurs in the absence of illumination. We demonstrate RESOLFT recordings both in chemically fixed and in living human cells using PENELOPE as a fusion protein.