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      A guide to super-resolution fluorescence microscopy

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          Abstract

          For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.

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          Most cited references 89

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          Far-field optical nanoscopy.

           Stefan Hell (2007)
          In 1873, Ernst Abbe discovered what was to become a well-known paradigm: the inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. However, for its most popular imaging mode, fluorescence microscopy, the diffraction barrier is crumbling. Here, I discuss the physical concepts that have pushed fluorescence microscopy to the nanoscale, once the prerogative of electron and scanning probe microscopes. Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.
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            Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.

            Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.
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              The fluorescent toolbox for assessing protein location and function.

              Advances in molecular biology, organic chemistry, and materials science have recently created several new classes of fluorescent probes for imaging in cell biology. Here we review the characteristic benefits and limitations of fluorescent probes to study proteins. The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small organic fluorescent dyes, nanocrystals ("quantum dots"), autofluorescent proteins, small genetic encoded tags that can be complexed with fluorochromes, and combinations of these probes are highlighted.
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                Author and article information

                Journal
                J Cell Biol
                J. Cell Biol
                jcb
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                26 July 2010
                : 190
                : 2
                : 165-175
                Affiliations
                [1 ]Department of Biology and Center for Integrated Protein Science, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany
                [2 ]King’s College London, Randall Division of Cell and Molecular Biophysics, New Hunt’s House, Guy’s Campus, London SE1 1UL, England, UK
                [3 ]Institute of Physical Chemistry, Friedrich-Schiller University Jena, 07743 Jena, Germany
                [4 ]Institute of Photonic Technology, 07745 Jena, Germany
                Author notes
                Correspondence to: Lothar Schermelleh: lothar.schermelleh@ 123456lmu.de ; Rainer Heintzmann: heintzmann@ 123456gmail.com ; or Heinrich Leonhardt: h.leonhardt@ 123456lmu.de
                Article
                201002018
                10.1083/jcb.201002018
                2918923
                20643879
                © 2010 Schermelleh et al.

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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