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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 111

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          Imaging intracellular fluorescent proteins at nanometer resolution.

          We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.
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            Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).

            We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.
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              Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.

              We propose a new type of scanning fluorescence microscope capable of resolving 35 nm in the far field. We overcome the diffraction resolution limit by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point-spread function. In contrast to near-field scanning optical microscopy, this method can produce three-dimensional images of translucent specimens.
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                Author and article information

                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
                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
                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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