Super-resolution fluorescence imaging with single molecules.

The ability to detect, image and localize single molecules optically with high spatial precision by their fluorescence enables an emergent class of super-resolution microscopy methods which have overcome the longstanding diffraction barrier for far-field light-focusing optics. Achieving spatial resolutions of 20-40nm or better in both fixed and living cells, these methods are currently being established as powerful tools for minimally-invasive spatiotemporal analysis of structural details in cellular processes which benefit from enhanced resolution. Briefly covering the basic principles, this short review then summarizes key recent developments and application examples of two-dimensional and three-dimensional (3D) multi-color techniques and faster time-lapse schemes. The prospects for quantitative imaging - in terms of improved ability to correct for dipole-emission-induced systematic localization errors and to provide accurate counts of molecular copy numbers within nanoscale cellular domains - are discussed.

[1]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[2]  W E Moerner,et al.  STED microscopy with optimized labeling density reveals 9-fold arrangement of a centriole protein. , 2012, Biophysical journal.

[3]  Lucien E. Weiss,et al.  Cellular Inclusion Bodies of Mutant Huntingtin Exon 1 Obscure Small Fibrillar Aggregate Species , 2012, Scientific Reports.

[4]  W. Webb,et al.  Precise nanometer localization analysis for individual fluorescent probes. , 2002, Biophysical journal.

[5]  S. Hell,et al.  Fluorescence nanoscopy by ground-state depletion and single-molecule return , 2008, Nature Methods.

[6]  S. Ram,et al.  Ultrahigh accuracy imaging modality for super-localization microscopy , 2013, Nature Methods.

[7]  Christian Eggeling,et al.  Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids , 2010, Proceedings of the National Academy of Sciences.

[8]  Christian Eggeling,et al.  Macromolecular-scale resolution in biological fluorescence microscopy. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[9]  J. J. Macklin,et al.  Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution , 2011, Proceedings of the National Academy of Sciences.

[10]  Harish Vashisth,et al.  Chromosome Organization by a Nucleoid-Associated Protein in Live Bacteria , 2013 .

[11]  W. E. Moerner,et al.  Optische Spektroskopie von einzelnen Dotierungsmolekülen in Festkörpern , 1993 .

[12]  Long Cai,et al.  Single cell systems biology by super-resolution imaging and combinatorial labeling , 2012, Nature Methods.

[13]  W E Moerner,et al.  Single-molecule mountains yield nanoscale cell images , 2006, Nature Methods.

[14]  S. Hell.  Toward fluorescence nanoscopy , 2003, Nature Biotechnology.

[15]  Thomas Basché,et al.  Optical Spectroscopy of Single Impurity Molecules in Solids , 1993 .

[16]  G. C. Rogers,et al.  Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization , 2012, Nature Cell Biology.

[17]  X. Zhuang,et al.  Superresolution Imaging of Chemical Synapses in the Brain , 2010, Neuron.

[18]  X. Xie,et al.  Single-Molecule Spectroscopy and Dynamics at Room Temperature , 1996 .

[19]  S. Hell,et al.  Properties of a 4Pi confocal fluorescence microscope , 1992 .

[20]  Thorsten Staudt,et al.  Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy. , 2011, Nano letters.

[21]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[22]  Matthew D Lew,et al.  The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision. , 2012, Applied physics letters.

[23]  A. Diaspro,et al.  Live-cell 3D super-resolution imaging in thick biological samples , 2011, Nature Methods.

[24]  W E Moerner,et al.  Enzymatic activation of nitro-aryl fluorogens in live bacterial cells for enzymatic turnover-activated localization microscopy† , 2013, Chemical science.

[25]  M. Heilemann,et al.  Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. , 2008, Angewandte Chemie.

[26]  Matthew D Lew,et al.  Rotational mobility of single molecules affects localization accuracy in super-resolution fluorescence microscopy. , 2013, Nano letters.

[27]  Matthew D. Lew,et al.  Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus , 2011, Proceedings of the National Academy of Sciences.

[28]  Ned S. Wingreen,et al.  Self-Organization of the Escherichia coli Chemotaxis Network Imaged with Super-Resolution Light Microscopy , 2009, PLoS biology.

[29]  Matthew D Lew,et al.  Simultaneous, accurate measurement of the 3D position and orientation of single molecules , 2012, Proceedings of the National Academy of Sciences.

[30]  Shigeki Iwanaga,et al.  Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores. , 2010, Journal of the American Chemical Society.

[31]  Julie S Biteen,et al.  Three-dimensional super-resolution imaging of the midplane protein FtsZ in live Caulobacter crescentus cells using astigmatism. , 2012, Chemphyschem : a European journal of chemical physics and physical chemistry.

[32]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[33]  S. Hell.  Far-field optical nanoscopy , 2010 .

[34]  Christian Eggeling,et al.  Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[35]  E. Betzig,et al.  Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics , 2008, Nature Methods.

[36]  Michael A Thompson,et al.  Super-resolution imaging of the nucleoid-associated protein HU in Caulobacter crescentus. , 2011, Biophysical journal.

[37]  W E Moerner,et al.  Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions. , 2013, Nano letters.

[38]  Fitnat H. Yildiz,et al.  Molecular Architecture and Assembly Principles of Vibrio cholerae Biofilms , 2012, Science.

[39]  W E Moerner,et al.  Sub-diffraction imaging of huntingtin protein aggregates by fluorescence blink-microscopy and atomic force microscopy. , 2011, Chemphyschem : a European journal of chemical physics and physical chemistry.

[40]  E. Abbe.  Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung , 1873 .

[41]  X. Zhuang,et al.  Evaluation of Fluorophores for Optimal Performance in Localization-Based Super-Resolution Imaging , 2012 .

[42]  S. Hell,et al.  Subdiffraction resolution in far-field fluorescence microscopy. , 1999, Optics letters.

[43]  Tobias M. P. Hartwich,et al.  Video-rate nanoscopy using sCMOS camera- specific single-molecule localization algorithms , 2013 .

[44]  Alexander Egner,et al.  Isotropic 3D Nanoscopy based on single emitter switching. , 2008, Optics express.

[45]  Hazen P. Babcock,et al.  Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton , 2011, Nature Methods.

[46]  Michael W. Davidson,et al.  Nanoscale architecture of integrin-based cell adhesions , 2010, Nature.

[47]  W. Moerner,et al.  Optical detection and spectroscopy of single molecules in a solid. , 1989, Physical review letters.

[48]  C. Bustamante,et al.  Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM) , 2012, Proceedings of the National Academy of Sciences.

[49]  S. Hess,et al.  Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples , 2008, Nature Methods.

[50]  Michael A Thompson,et al.  Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP , 2008, Nature Methods.

[51]  Prabuddha Sengupta,et al.  Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis , 2011, Nature Methods.

[52]  Bernd Rieger,et al.  Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution , 2012, Journal of Cell Science.

[53]  Christian Eggeling,et al.  Diffraction-unlimited all-optical imaging and writing with a photochromic GFP , 2011, Nature.

[54]  P. Annibale,et al.  Quantitative Photo Activated Localization Microscopy: Unraveling the Effects of Photoblinking , 2011, PloS one.

[55]  Robert J. Chichester,et al.  Single Molecules Observed by Near-Field Scanning Optical Microscopy , 1993, Science.

[56]  Andrew G. York,et al.  Confined Activation and Subdiffractive Localization Enables Whole-Cell PALM with Genetically Expressed Probes , 2011, Nature Methods.

[57]  R. Hochstrasser,et al.  Wide-field subdiffraction imaging by accumulated binding of diffusing probes , 2006, Proceedings of the National Academy of Sciences.

[58]  S. Hell,et al.  Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores , 2011, Nature Methods.

[59]  Jonas Fölling,et al.  Imaging nanometer-sized α-synuclein aggregates by superresolution fluorescence localization microscopy. , 2012, Biophysical journal.

[60]  Shigeki Iwanaga,et al.  Fluorescent saxitoxins for live cell imaging of single voltage-gated sodium ion channels beyond the optical diffraction limit. , 2012, Chemistry & biology.

[61]  J. Lippincott-Schwartz,et al.  High-density mapping of single-molecule trajectories with photoactivated localization microscopy , 2008, Nature Methods.

[62]  M. Gustafsson.  Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[63]  H. Ewers,et al.  A simple, versatile method for GFP-based super-resolution microscopy via nanobodies , 2012, Nature Methods.

[64]  E. Ullian,et al.  Afadin, A Ras/Rap Effector That Controls Cadherin Function, Promotes Spine and Excitatory Synapse Density in the Hippocampus , 2012, The Journal of Neuroscience.

[65]  Chenglong Xia,et al.  Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes , 2012, Proceedings of the National Academy of Sciences.

[66]  Matthew D. Lew,et al.  Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane. , 2010, Applied physics letters.

[67]  X. Zhuang,et al.  Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in Axons , 2013, Science.

[68]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[69]  W E Moerner,et al.  New directions in single-molecule imaging and analysis , 2007, Proceedings of the National Academy of Sciences.

[70]  J. Lippincott-Schwartz,et al.  Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure , 2009, Proceedings of the National Academy of Sciences.

[71]  Mark Bates,et al.  Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy , 2008, Science.

[72]  Dylan T Burnette,et al.  Bayesian localisation microscopy reveals nanoscale podosome dynamics , 2011, Nature Methods.

[73]  Suliana Manley,et al.  Live‐Cell dSTORM of Cellular DNA Based on Direct DNA Labeling , 2012, Chembiochem : a European journal of chemical biology.

[74]  M. Gustafsson.  Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[75]  C. Dobson,et al.  In situ measurements of the formation and morphology of intracellular β-amyloid fibrils by super-resolution fluorescence imaging. , 2011, Journal of the American Chemical Society.

[76]  S. Ram,et al.  Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions , 2004, IEEE Transactions on NanoBioscience.

[77]  W. Moerner,et al.  Illuminating single molecules in condensed matter. , 1999, Science.

[78]  Samuel J. Lord,et al.  Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function , 2009, Proceedings of the National Academy of Sciences.

[79]  Helmut Grubmüller,et al.  How SNARE molecules mediate membrane fusion: recent insights from molecular simulations. , 2012, Current opinion in structural biology.

[80]  S. Hell.  Microscopy and its focal switch , 2008, Nature Methods.

[81]  Lei Zhu,et al.  Faster STORM using compressed sensing , 2012, Nature Methods.

[82]  S. Holden,et al.  DAOSTORM: an algorithm for high- density super-resolution microscopy , 2011, Nature Methods.

[83]  N. Bobroff.  Position measurement with a resolution and noise‐limited instrument , 1986 .

[84]  Shu Jia,et al.  Ultra-bright Photoactivatable Fluorophores Created by Reductive Caging , 2012, Nature Methods.

[85]  R. Tsien,et al.  On/off blinking and switching behaviour of single molecules of green fluorescent protein , 1997, Nature.

[86]  W. E. Moerner,et al.  Localizing and tracking single nanoscale emitters in three dimensions with high spatiotemporal resolution using a double-helix point spread function. , 2010, Nano letters.

[87]  W. E. Moerner,et al.  Single-Molecule Optical Spectroscopy and Imaging: From Early Steps to Recent Advances , 2010 .

[88]  Zemer Gitai,et al.  Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[89]  L. Shapiro,et al.  A spindle-like apparatus guides bacterial chromosome segregation , 2010, Nature Cell Biology.

[90]  Matthew D Lew,et al.  Corkscrew point spread function for far-field three-dimensional nanoscale localization of pointlike objects. , 2011, Optics letters.

[91]  X. Zhuang,et al.  Fast three-dimensional super-resolution imaging of live cells , 2011, Nature Methods.

[92]  Jan Vogelsang,et al.  Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy , 2009, Proceedings of the National Academy of Sciences.

[93]  Fitnat H. Yildiz,et al.  Superresolution Imaging of Intact Microbial Communities Reveals Molecular Architecture of Biofilm Development and Bacterial Organization , 2011 .

[94]  Andrew D Ellington,et al.  Aptamers as potential tools for super-resolution microscopy , 2012, Nature Methods.