Fast Sted Microscopy with Continuous Wave Fiber Lasers References and Links

We report on fast beam-scanning stimulated-emission-depletion (STED) microscopy in the visible range using for resolution enhancement compact, low cost and turn-key continuous wave (CW) fiber lasers emitting at 592 nm. Spatial resolutions of 35 to 65 nm in the focal plane are shown for various samples including fluorescent nanoparticles, immuno-stained cells with a non-exhaustive selection of 5 commonly used organic fluorescent markers, and living cells expressing the yellow fluorescent protein Citrine. The potential of the straightforward combination of CW-STED and fast beam scanning is illustrated in a movie of the endoplasmic reticulum (ER) of a living cell, composed of 100 frames (6 microm x 12 microm), each of them acquired in a time shorter than 0.2 s.

[1]  S. Hell,et al.  Subdiffraction resolution in far-field fluorescence microscopy. , 1999, Optics letters.

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

[3]  S. Hell,et al.  STED microscopy with a supercontinuum laser source. , 2008, Optics express.

[4]  Christian Eggeling,et al.  Triplet-relaxation microscopy with bunched pulsed excitation , 2009, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[5]  T. Bonhoeffer,et al.  Live-cell imaging of dendritic spines by STED microscopy , 2008, Proceedings of the National Academy of Sciences.

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

[7]  R. Tsien,et al.  Reducing the Environmental Sensitivity of Yellow Fluorescent Protein , 2001, The Journal of Biological Chemistry.

[8]  Christian Eggeling,et al.  STED microscopy reveals crystal colour centres with nanometric resolution. , 2009 .

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

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

[11]  S. Hell,et al.  Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy , 2007, Neuroscience.

[12]  S. Hell,et al.  Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell , 2008, Proceedings of the National Academy of Sciences.

[13]  Alexander Egner,et al.  Tuning of synapse number, structure and function in the cochlea , 2009, Nature Neuroscience.

[14]  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 .

[15]  Christian Eggeling,et al.  Major signal increase in fluorescence microscopy through dark-state relaxation , 2007, Nature Methods.

[16]  S. Hell,et al.  Direct observation of the nanoscale dynamics of membrane lipids in a living cell , 2009, Nature.

[17]  Volker Westphal,et al.  A STED microscope aligned by design. , 2009, Optics express.

[18]  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.

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

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