Two-color RESOLFT nanoscopy with green and red fluorescent photochromic proteins.

Up to now, all demonstrations of reversible saturable optical fluorescence transitions (RESOLFT) superresolution microscopy of living cells have relied on the use of reversibly switchable fluorescent proteins (RSFP) emitting in the green spectral range. Here we show RESOLFT imaging with rsCherryRev1.4, a new red-emitting RSFP enabling a spatial resolution up to four times higher than the diffraction barrier. By co-expressing green and red RSFPs in living cells we demonstrate two-color RESOLFT imaging both for single ("donut") beam scanning and for parallelized versions of RESOLFT nanoscopy where an array of >23,000 "donut-like" minima are scanned simultaneously.

[1]  S. Hell.  Toward fluorescence nanoscopy , 2003, Nature Biotechnology.

[2]  S. Hell.  Microscopy and its focal switch , 2008, Nature Methods.

[3]  Stefan W. Hell,et al.  Supporting Online Material Materials and Methods Figs. S1 to S9 Tables S1 and S2 References Video-rate Far-field Optical Nanoscopy Dissects Synaptic Vesicle Movement , 2022 .

[4]  Christian Eggeling,et al.  Nanoscopy of Living Brain Slices with Low Light Levels , 2012, Neuron.

[5]  Christian Eggeling,et al.  rsEGFP2 enables fast RESOLFT nanoscopy of living cells , 2012, eLife.

[6]  Michael A Thompson,et al.  Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP , 2008, Nature Methods.

[7]  Peter Dedecker,et al.  Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching , 2006, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[8]  S. Hell,et al.  Fluorescence nanoscopy by ground-state depletion and single-molecule return , 2008, Nature Methods.

[9]  R. Quatrano.  Genomics , 1998, Plant Cell.

[10]  Christian Eggeling,et al.  A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching , 2011, Nature Biotechnology.

[11]  Marcus Dyba,et al.  Immunofluorescence stimulated emission depletion microscopy , 2003, Nature Biotechnology.

[12]  Konstantin A Lukyanov,et al.  Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET. , 2010, Chemistry & biology.

[13]  J. Rogers,et al.  hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes , 2007, Genomics.

[14]  R. Rosenfeld.  Nature , 2009, Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery.

[15]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[16]  Christian Eggeling,et al.  Diffraction-unlimited all-optical imaging and writing with a photochromic GFP , 2011, Nature.

[17]  S. Hell,et al.  Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Christian Eggeling,et al.  Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Stefan W. Hell,et al.  Nanoscopy in a Living Mouse Brain , 2012, Science.

[20]  Vladislav V Verkhusha,et al.  Chromophore transformations in red fluorescent proteins. , 2012, Chemical reviews.

[21]  Christian Eggeling,et al.  Nanoscopy with more than 100,000 'doughnuts' , 2013, Nature Methods.

[22]  R. Tsien,et al.  On/off blinking and switching behaviour of single molecules of green fluorescent protein , 1997, Nature.

[23]  S. Hell,et al.  Imaging and writing at the nanoscale with focused visible light through saturable optical transitions , 2003 .

[24]  Christian Eggeling,et al.  Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy. , 2008, Biophysical journal.

[25]  Philip Tinnefeld,et al.  Fluoreszenzmikroskopie unterhalb der optischen Auflösungsgrenze mit konventionellen Fluoreszenzsonden , 2008 .

[26]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[27]  E. Abbe.  Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung , 1873 .

[28]  Christian Eggeling,et al.  1.8 A bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants. , 2007, The Biochemical journal.

[29]  D. Wilkin,et al.  Neuron , 2001, Brain Research.

[30]  M. Gustafsson.  Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[32]  M. Heilemann,et al.  Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. , 2008, Angewandte Chemie.

[33]  R. Heintzmann,et al.  Saturated patterned excitation microscopy--a concept for optical resolution improvement. , 2002, Journal of the Optical Society of America. A, Optics, image science, and vision.