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      SOFI Simulation Tool: A Software Package for Simulating and Testing Super-Resolution Optical Fluctuation Imaging

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          Abstract

          Super-resolution optical fluctuation imaging (SOFI) allows one to perform sub-diffraction fluorescence microscopy of living cells. By analyzing the acquired image sequence with an advanced correlation method, i.e. a high-order cross-cumulant analysis, super-resolution in all three spatial dimensions can be achieved. Here we introduce a software tool for a simple qualitative comparison of SOFI images under simulated conditions considering parameters of the microscope setup and essential properties of the biological sample. This tool incorporates SOFI and STORM algorithms, displays and describes the SOFI image processing steps in a tutorial-like fashion. Fast testing of various parameters simplifies the parameter optimization prior to experimental work. The performance of the simulation tool is demonstrated by comparing simulated results with experimentally acquired data.

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          Most cited references7

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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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            Studies on the propagation in vitro of poliomyelitis viruses. IV. Viral multiplication in a stable strain of human malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix.

            The cells of a human epithelial cancer cultivated en masse have been shown to support the multiplication of all three types of poliomyelitis virus. These cells (strain HeLa of Gey) have been maintained in vitro since their derivation from an epidermoid carcinoma of the cervix in February, 1951. As the virus multiplied it caused in from 12 to 96 hours degeneration and destruction of the cancer cells. The specific destructive effect of the virus was prevented by adding homotypic antibody to the cultures but not by adding heterotypic antibodies. Methods for the preparation of large numbers of replicate cultures with suspensions of strain HeLa cells were described. The cells in suspension were readily quantitated by direct counts in a hemocytometer. A synthetic solution that maintains cellular viability was employed for viral propagation. The experimental results demonstrate the usefulness of strain HeLa cells for (a) the quantitation of poliomyelitis virus, (b) the measurement of poliomyelitis antibodies, and (c) the production of virus.
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              A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching.

              Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of ∼365 nm and ∼405 nm, respectively, whereas fluorescence is elicited at ∼515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                2016
                1 September 2016
                : 11
                : 9
                : e0161602
                Affiliations
                [1 ]Laboratoire d’Optique Biomédicale, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
                [2 ]Department of Chemistry, University of Leuven, Celestijnenlaan, Heverlee, Belgium
                [3 ]Department of Radioelectronics, Faculty of Electrical Engineering, Czech Technical University in Prague, Prague, Czech Republic
                University of California Berkeley, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: T. Lasser.

                • Data curation: AG AS.

                • Formal analysis: SG ML.

                • Funding acquisition: T. Lasser.

                • Investigation: AG T. Lukes AS.

                • Methodology: AG T. Lukes T. Lasser.

                • Project administration: T. Lasser JH.

                • Resources: AS T. Lasser.

                • Software: AG T. Lukes ML.

                • Supervision: T. Lasser.

                • Validation: AG T. Lukes WV PD.

                • Visualization: AG T. Lukes T. Lasser.

                • Writing original draft: AG T. Lukes T. Lasser.

                • Writing review & editing: AG T. Lukes T. Lasser PD JH.

                [¤]

                Current address: Abteilung NanoBiophotonik, Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany

                Article
                PONE-D-16-20236
                10.1371/journal.pone.0161602
                5008722
                27583365
                0074bdb2-1071-44ce-a025-33c04a9616eb
                © 2016 Girsault et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 19 May 2016
                : 8 August 2016
                Page count
                Figures: 7, Tables: 0, Pages: 13
                Funding
                Funded by: Swiss National Science Foundation (SNSF)
                Award ID: 200020-159945/1
                Award Recipient :
                Funded by: Swiss National Science Foundation (SNSF)
                Award ID: 205321-138305/1
                Award Recipient :
                Funded by: SCIEX scholarship
                Award ID: 13.183
                Award Recipient :
                This research was supported by the Swiss National Science Foundation (SNSF, http://www.snf.ch/en) under grants 200020-159945/1 and 205321-138305/1. Tomas Lukes acknowledges a SCIEX scholarship (13.183).
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Biochemical Simulations
                Biology and Life Sciences
                Computational Biology
                Biochemical Simulations
                Research and Analysis Methods
                Imaging Techniques
                Fluorescence Imaging
                Physical Sciences
                Mathematics
                Applied Mathematics
                Algorithms
                Research and Analysis Methods
                Simulation and Modeling
                Algorithms
                Research and analysis methods
                Biological cultures
                Cell lines
                HeLa cells
                Research and analysis methods
                Biological cultures
                Cell cultures
                Cultured tumor cells
                HeLa cells
                Research and Analysis Methods
                Simulation and Modeling
                Physical Sciences
                Chemistry
                Chemical Reactions
                Bleaching
                Research and Analysis Methods
                Microscopy
                Light Microscopy
                Fluorescence Microscopy
                Engineering and Technology
                Equipment
                Optical Equipment
                Cameras
                Custom metadata
                Software package and a user guide are freely available at http://lob.epfl.ch/sofitool/. The software is now freely available also at GitHub public repository according to Plos One recommendation for software sharing. https://github.com/lob-epfl/sofitool.

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                Uncategorized

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