Nanoscopy with more than 100,000 'doughnuts'

We show that nanoscopy based on the principle called RESOLFT (reversible saturable optical fluorescence transitions) or nonlinear structured illumination can be effectively parallelized using two incoherently superimposed orthogonal standing light waves. The intensity minima of the resulting pattern act as 'doughnuts', providing isotropic resolution in the focal plane and making pattern rotation redundant. We super-resolved living cells in 120 μm × 100 μm–sized fields of view in <1 s using 116,000 such doughnuts.

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

[2]  M. Gustafsson,et al.  Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. , 2008, Biophysical journal.

[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.  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.

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

[6]  J. J. Macklin,et al.  Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution , 2011, Proceedings of the National Academy of Sciences.

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

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

[9]  Johann Engelhardt,et al.  Parallelized STED fluorescence nanoscopy. , 2011, Optics express.

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

[11]  M. Gustafsson Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

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

[13]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[14]  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.

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

[16]  A. Stemmer,et al.  True optical resolution beyond the Rayleigh limit achieved by standing wave illumination. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[17]  S. Hell,et al.  Wide‐field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching , 2007, Microscopy research and technique.

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

[19]  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.

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