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      Video-rate nanoscopy enabled by sCMOS camera-specific single-molecule localization algorithms

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          Abstract

          Newly developed scientific complementary metal–oxide–semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition in single-molecule switching nanoscopy (SMSN) while simultaneously increasing the effective quantum efficiency. However, sCMOS-intrinsic pixel-dependent readout noise substantially reduces the localization precision and introduces localization artifacts. Here we present algorithms that overcome these limitations and provide unbiased, precise localization of single molecules at the theoretical limit. In combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at up to 32 reconstructed images/second (recorded at 1,600–3,200 camera frames/second) in both fixed and living cells.

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          High-density mapping of single-molecule trajectories with photoactivated localization microscopy.

          Suliana Manley, Jennifer Gillette, George Patterson … (2008)
          We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.
          Article 0 comments Cited 384 times – based on 0 reviews     Review now
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            A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins.

            Gražvydas Lukinavičius, Keitaro Umezawa, Nicolas Olivier … (2013)
            The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proved to be extremely difficult. Here, we introduce a biocompatible near-infrared silicon-rhodamine probe that can be coupled specifically to proteins using different labelling techniques. Importantly, its high permeability and fluorogenic character permit the imaging of proteins in living cells and tissues, and its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.
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              Optimized localization-analysis for single-molecule tracking and super-resolution microscopy

              Kim Mortensen, L Stirling Churchman, James Spudich … (2010)
              We optimally localize isolated fluorescent beads and molecules imaged as diffraction-limited spots, determine the orientation of molecules, and present reliable formulae for the precisions of various localization methods. For beads, theory and experimental data both show that unweighted least-squares fitting of a Gaussian squanders one third of the available information, a popular formula for its precision exaggerates beyond Fisher's information limit, and weighted least-squares may do worse, while maximum likelihood fitting is practically optimal.
              Article 0 comments Cited 258 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-journal-id): 101215604
                Journal ID (pubmed-jr-id): 32338
                Journal ID (nlm-ta): Nat Methods
                Journal ID (iso-abbrev): Nat. Methods
                Title: Nature methods
                ISSN (Print): 1548-7091
                ISSN (Electronic): 1548-7105
                Publication date Nihms-submitted: 23 May 2013
                Publication date (Electronic): 26 May 2013
                Publication date (Print): July 2013
                Publication date PMC-release: 26 November 2013
                Volume: 10
                Issue: 7
                Electronic Location Identifier: 10.1038/nmeth.2488
                Affiliations
                [1 ]Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA
                [2 ]Department of Biophysical Chemistry, University of Heidelberg, 69120 Heidelberg, Germany
                [3 ]Department of New Materials and Biosystems, Max Planck Institute for Intelligent Systems, 70569 Stuttgart, Germany
                [4 ]Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA
                [5 ]Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, CT 06520, USA
                [6 ]Yale College, Yale University, New Haven, CT 06520, USA
                [7 ]Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06520, USA
                [8 ]National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, FL 32310, USA
                Author notes
                [* ]Correspondence to: Joerg Bewersdorf joerg.bewersdorf@ 123456yale.edu
                [9]

                These authors contributed equally to this work

                Article
                Manuscript ID: NIHMS475969
                DOI: 10.1038/nmeth.2488
                PMC ID: 3696415
                PubMed ID: 23708387
                SO-VID: 133bdc25-e79a-49d1-a9ef-7cf3d0047ddf
                License:

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Funding
                Funded by: National Cancer Institute : NCI
                Award ID: R01 CA098727 || CA
                Categories
                Subject: Article

                ScienceOpen disciplines: Life sciences
                Data availability:
                ScienceOpen disciplines: Life sciences

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