Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters

We demonstrate nanoscale resolution in far-field fluorescence microscopy using reversible photoswitching and localization of individual fluorophores at comparatively fast recording speeds and from the interior of intact cells. These advancements have become possible by asynchronously recording the photon bursts of individual molecular switching cycles. We present images from the microtubular network of an intact mammalian cell with a resolution of 40 nm.

[1]  J. Högbom,et al.  APERTURE SYNTHESIS WITH A NON-REGULAR DISTRIBUTION OF INTERFEROMETER BASELINES. Commentary , 1974 .

[2]  F. Pinaud,et al.  Ultrahigh-resolution multicolor colocalization of single fluorescent probes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

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

[4]  Stefan W. Hell,et al.  Improvement of lateral resolution in far-field fluorescence light microscopy by using two-photon excitation with offset beams , 1994 .

[5]  J. Slot,et al.  Improving structural integrity of cryosections for immunogold labeling , 1996, Histochemistry and Cell Biology.

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

[7]  S. Hell,et al.  Two- and multiphoton detection as an imaging mode and means of increasing the resolution in far-field light microscopy: A study based on photon-optics , 1995 .

[8]  A. Miyawaki,et al.  Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting , 2004, Science.

[9]  X Michalet,et al.  Ultrahigh-resolution colocalization of spectrally separable point-like fluorescent probes. , 2001, Methods.

[10]  W. Webb,et al.  Precise nanometer localization analysis for individual fluorescent probes. , 2002, Biophysical journal.

[11]  N. Bobroff Position measurement with a resolution and noise‐limited instrument , 1986 .

[12]  W E Moerner,et al.  Single-molecule mountains yield nanoscale cell images , 2006, Nature Methods.

[13]  E. Betzig,et al.  Proposed method for molecular optical imaging. , 1995, Optics letters.

[14]  G. von Heijne,et al.  Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli , 2001, FEBS letters.

[15]  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.

[16]  A. Egner,et al.  Resolution of λ /10 in fluorescence microscopy using fast single molecule photo-switching , 2007 .

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

[18]  S. Hell Far-Field Optical Nanoscopy , 2007, Science.

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

[20]  K Weber,et al.  Visualization of a system of filaments 7-10 nm thick in cultured cells of an epithelioid line (Pt K2) by immunofluorescence microscopy. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[21]  K. Tokuyasu A TECHNIQUE FOR ULTRACRYOTOMY OF CELL SUSPENSIONS AND TISSUES , 1973, The Journal of cell biology.

[22]  K. Tokuyasu Immunochemistry on ultrathin frozen sections , 1980, The Histochemical Journal.

[23]  H Schindler,et al.  Imaging of single molecule diffusion. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

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