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      Fluorescent microscopy beyond diffraction limits using speckle illumination and joint support recovery

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          Abstract

          Structured illumination microscopy (SIM) breaks the optical diffraction limit by illuminating a sample with a series of line-patterned light. Recently, in order to alleviate the requirement of precise knowledge of illumination patterns, structured illumination microscopy techniques using speckle patterns have been proposed. However, these methods require stringent assumptions of the speckle statistics: for example, speckle patterns should be nearly incoherent or their temporal average should be roughly homogeneous. Here, we present a novel speckle illumination microscopy technique that overcomes the diffraction limit by exploiting the minimal requirement that is common for all the existing super-resolution microscopy, i.e. that the fluorophore locations do not vary during the acquisition time. Using numerical and real experiments, we demonstrate that the proposed method can improve the resolution up to threefold. Because our proposed method succeeds for standard fluorescence probes and experimental protocols, it can be applied in routine biological experiments.

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          Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution.

          Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arises from saturation of the excited state. This method can be used in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.
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            Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization.

            Myosin V is a dimeric molecular motor that moves processively on actin, with the center of mass moving approximately 37 nanometers for each adenosine triphosphate hydrolyzed. We have labeled myosin V with a single fluorophore at different positions in the light-chain domain and measured the step size with a standard deviation of <1.5 nanometers, with 0.5-second temporal resolution, and observation times of minutes. The step size alternates between 37 + 2x nm and 37 - 2x, where x is the distance along the direction of motion between the dye and the midpoint between the two heads. These results strongly support a hand-over-hand model of motility, not an inchworm model.
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              Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI).

              Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                25 June 2013
                2013
                : 3
                : 2075
                Affiliations
                [1 ]Department of Bio and Brain Engineering , KAIST, Daejeon, Korea
                [2 ]Samsung Advanced Institute of Technology , Suwon, Korea
                [3 ]These authors contributed equally to this work.
                Author notes
                Article
                srep02075
                10.1038/srep02075
                3691569
                23797902
                60bcdbbc-94c4-4832-acd2-d3f1a2e2be90
                Copyright © 2013, Macmillan Publishers Limited. All rights reserved

                This work is licensed under a Creative Commons Attribution-NonCommercial-ShareALike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/

                History
                : 25 March 2013
                : 10 June 2013
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