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      Quantitative Super-Resolution Imaging with qPAINT using Transient Binding Analysis

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          Abstract

          Current super-resolution techniques offer unprecedented spatial resolution, but quantitative counting of spatially unresolvable molecules remains challenging. Here, we use the programmable and specific binding of dye-labeled DNA probes to count integer numbers of targets. This method, called quantitative Points Accumulation In Nanoscale Topography (qPAINT), avoids the challenging task of analyzing the environmentally sensitive hard-to-predict photophysics of dyes, and enables robust counting by analyzing the predictable binding kinetics of dye-labeled DNA probes. We benchmarked qPAINT in vitro and in situ by counting strands on DNA nanostructures, Nup98 protein clusters in the nuclear pore complex, Bruchpilot proteins in Drosophila, and finally the number of fluorescence in situ hybridization probes on single mRNA targets in fixed cells. We achieved high accuracy (~98–99 %), high precision (~80–95 %), and multiplexed detection over a large dynamic range.

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          Most cited references29

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          Multiplexed 3D Cellular Super-Resolution Imaging with DNA-PAINT and Exchange-PAINT

          While super-resolution fluorescence microscopy is a powerful tool for biological research, obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed 3D super-resolution imaging inside fixed cells and achieve sub-10 nm spatial resolution in vitro using synthetic DNA structures. We also report a novel approach for multiplexing (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-“color” super-resolution imaging in vitro on synthetic DNA structures and four-“color” imaging of proteins in a fixed cell.
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            Wide-field subdiffraction imaging by accumulated binding of diffusing probes.

            A method is introduced for subdiffraction imaging that accumulates points by collisional flux. It is based on targeting the surface of objects by fluorescent probes diffusing in the solution. Because the flux of probes at the object is essentially constant over long time periods, the examination of an almost unlimited number of individual probe molecules becomes possible. Each probe that hits the object and that becomes immobilized is located with high precision by replacing its point-spread function by a point at its centroid. Images of lipid bilayers, contours of these bilayers, and large unilamellar vesicles are shown. A spatial resolution of approximately 25 nm is readily achieved. The ability of the method to effect rapid nanoscale imaging and spatial resolution below Rayleigh criterion and without the necessity for labeling with fluorescent probes is proven.
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              Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami.

              DNA origami is a powerful method for the programmable assembly of nanoscale molecular structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resolution becomes increasingly important. We present a single-molecule assay for the study of binding and unbinding kinetics on DNA origami. We find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, we exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topography) imaging with <30 nm resolution. The method is demonstrated for flat monomeric DNA structures as well as multimeric, ribbon-like DNA structures.
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                Author and article information

                Journal
                101215604
                32338
                Nat Methods
                Nat. Methods
                Nature methods
                1548-7091
                1548-7105
                11 May 2016
                28 March 2016
                May 2016
                28 September 2016
                : 13
                : 5
                : 439-442
                Affiliations
                [1 ]Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA
                [2 ]Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA
                [3 ]Program in Biophysics, Harvard University, Boston, Massachusetts, USA
                [4 ]Rosenstiel Basic Medical Sciences Research Center, Department of Biology, Brandeis University, Waltham, Massachusetts, USA
                Author notes
                Correspondence should be addressed to P.Y. ( py@ 123456hms.harvard.edu )
                [5]

                Present addresses: Max Planck Institue of Biochemistry and Ludwig Maximilian University, Munich, Germany (R.J. and J.B.W.); Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Jakkur, Bangalore, India (S.S.A).

                [6]

                These authors contributed equally to this work.

                Article
                NIHMS763946
                10.1038/nmeth.3804
                4941813
                27018580
                c8cfe6bb-4968-44c1-a2b4-e136795a0c65

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