STED microscopy with a supercontinuum laser source.

We report on a straightforward yet powerful implementation of stimulated emission depletion (STED) fluorescence microscopy providing subdiffraction resolution in the far-field. Utilizing the same super-continuum pulsed laser source both for excitation and STED, this implementation of STED microscopy avoids elaborate preparations of laser pulses and conveniently provides multicolor imaging. Operating at pulse repetition rates around 1 MHz, it also affords reduced photobleaching rates by allowing the fluorophore to relax from excitable metastable dark states involved in photodegradation. The imaging of dense nanoparticles and of the microtubular network of mammalian cells evidences a spatial resolution of 30-50 nm in the focal plane, i.e. by a factor of 8-9 beyond the diffraction barrier.

[1]  Andreas Schönle,et al.  Resolution scaling in STED microscopy. , 2008, Optics express.

[2]  M. Neil,et al.  Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging. , 2008, Optics letters.

[3]  S. Hell,et al.  STED microscopy with continuous wave beams , 2007, Nature Methods.

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

[5]  Andreas Schönle,et al.  Efficient fluorescence inhibition patterns for RESOLFT microscopy. , 2007, Optics express.

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

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

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

[9]  Christian Eggeling,et al.  Macromolecular-scale resolution in biological fluorescence microscopy. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Grover A Swartzlander,et al.  Achromatic optical vortex lens. , 2006, Optics letters.

[11]  S. Hell,et al.  STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis , 2006, Nature.

[12]  Toby D M Bell,et al.  Characterizing the fluorescence intermittency and photobleaching kinetics of dye molecules immobilized on a glass surface. , 2006, The journal of physical chemistry. A.

[13]  Volker Westphal,et al.  Nanoscale resolution in the focal plane of an optical microscope. , 2005, Physical review letters.

[14]  Michel Orrit,et al.  Photobleaching of Rhodamine 6G in Poly(vinyl alcohol) at the Ensemble and Single-Molecule Levels , 2004 .

[15]  P. Russell,et al.  Photonic Crystal Fibers , 2003, Science.

[16]  D. Bout,et al.  Single molecule photobleaching: increasing photon yield and survival time through suppression of two-step photolysis , 2002 .

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

[19]  A. Stentz,et al.  Visible continuum generation in air–silica microstructure optical fibers with anomalous dispersion at 800 nm , 2000 .

[20]  K. Weber,et al.  Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulin-containing structures. , 1982, Methods in cell biology.