1. Single-molecule localization microscopy
- Author
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Markus Sauer, Florian Schueder, Melike Lakadamyali, Gerti Beliu, Melina Theoni Gyparaki, Suliana Manley, Ralf Jungmann, Juliette Griffié, Mickaël Lelek, Christophe Zimmer, Imagerie et Modélisation - Imaging and Modeling, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), University of Pennsylvania, University of Würzburg = Universität Würzburg, Ludwig Maximilian University [Munich] (LMU), Max-Planck-Institut für Biochemie = Max Planck Institute of Biochemistry (MPIB), Max-Planck-Gesellschaft, Ecole Polytechnique Fédérale de Lausanne (EPFL), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), C.Z. acknowledges funding by Institut Pasteur, Fondation pour la Recherche Médicale (grant DEQ 20150331762), Région Ile de France, Agence Nationale de la Recherche and Investissement d’Avenir grant ANR-16-CONV-0005. M.La. acknowledges funding from the National Institutes of Health/National Institutes of General Medical Sciences (NIH/NIGMS) under grant RO1 GM133842-01. G.B. and M.S. acknowledge funding by the German Research Foundation (DFG) (SA829/19-1) and the European Regional Development Fund (EFRE project ‘Center for Personalized Molecular Immunotherapy’). F.S. and R.J. acknowledge support by the DFG through SFB1032 (project A11) and the Max Planck Society. J.G. and S.M. acknowledge funding by the European Union’s H2020 programme under the Marie Skłodowska-Curie grant BALTIC (to J.G.) and ERC Piko (to S.M.)., The authors apologize to the authors of numerous papers that could not be cited owing to limited space. M.Le. and C.Z. thank B. Lelandais for excellent comments on the manuscript and M. Singh for acquiring the image shown in Fig. 3b., ANR-16-CONV-0005,INCEPTION,Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs(2016), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), University of Pennsylvania [Philadelphia], Max Planck Institute of Biochemistry (MPIB), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPC)
- Subjects
Single molecule localization ,light-microscopy ,Computer science ,business.industry ,General Medicine ,Article ,General Biochemistry, Genetics and Molecular Biology ,optical reconstruction microscopy ,diffraction-limit ,Fluorescent labelling ,3-dimensional superresolution ,live-cell ,correlative superresolution fluorescence ,Microscopy ,Time course ,Image acquisition ,colocalization analysis ,[SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie ,Computer vision ,Artificial intelligence ,business ,living cells ,intramolecular spirocyclization ,Image resolution ,electron-microscopy - Abstract
Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM. This Primer explains the central concepts of single-molecule localization microscopy (SMLM) before discussing experimental considerations regarding fluorophores, optics and data acquisition, processing and analysis. The Primer further describes recent high-impact discoveries made by SMLM techniques and concludes by discussing emerging methodologies.
- Published
- 2021