Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

[1]  P. Janmey,et al.  Counterion-mediated cluster formation by polyphosphoinositides. , 2014, Chemistry and physics of lipids.

[2]  Titiwat Sungkaworn,et al.  Single-molecule analysis of fluorescently labeled G-protein–coupled receptors reveals complexes with distinct dynamics and organization , 2012, Proceedings of the National Academy of Sciences.

[3]  P. Haggie,et al.  Confinement of β1- and β2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae , 2011, Molecular biology of the cell.

[4]  K. Jacobson,et al.  Single-particle tracking: applications to membrane dynamics. , 1997, Annual review of biophysics and biomolecular structure.

[5]  W. Webb,et al.  Fluorescence probe partitioning between Lo/Ld phases in lipid membranes. , 2007, Biochimica et biophysica acta.

[6]  M. Poo,et al.  Rates of membrane-associated reactions: reduction of dimensionality revisited , 1986, The Journal of cell biology.

[7]  Michelle D. Wang,et al.  Reduction-of-dimensionality kinetics at reaction-limited cell surface receptors. , 1994, Biophysical journal.

[8]  Xianlin Han,et al.  Lipidomics Analyses Reveal Temporal and Spatial Lipid Organization and Uncover Daily Oscillations in Intracellular Organelles. , 2016, Molecular cell.

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

[10]  John W Sedat,et al.  Phase retrieval for high-numerical-aperture optical systems. , 2003, Optics letters.

[11]  P. Roller,et al.  Formaldehyde fixation. , 1985, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[12]  P. Schwille,et al.  Photoconversion of Bodipy‐Labeled Lipid Analogues , 2013, Chembiochem : a European journal of chemical biology.

[13]  M. Ameloot,et al.  Membrane distribution of the glycine receptor α3 studied by optical super-resolution microscopy , 2014, Histochemistry and Cell Biology.

[14]  J. Zimmerberg,et al.  Lipid polymorphisms and membrane shape. , 2011, Cold Spring Harbor perspectives in biology.

[15]  Michael P. Sheetz,et al.  Cell control by membrane–cytoskeleton adhesion , 2001, Nature Reviews Molecular Cell Biology.

[16]  Mark M. Davis,et al.  T-cell-antigen recognition and the immunological synapse , 2003, Nature Reviews Immunology.

[17]  Michael W. Davidson,et al.  Video-rate nanoscopy enabled by sCMOS camera-specific single-molecule localization algorithms , 2013, Nature Methods.

[18]  S. E. Irvine,et al.  Fast Sted Microscopy with Continuous Wave Fiber Lasers References and Links , 2022 .

[19]  Peter J. Verveer,et al.  Chemically Induced Photoswitching of Fluorescent Probes—A General Concept for Super-Resolution Microscopy , 2011, Molecules.

[20]  Enrico Gratton,et al.  Mapping the Number of Molecules and Brightness in the Laser Scanning Microscope , 2007, Biophysical journal.

[21]  M. Davidson,et al.  Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics , 2015, Science.

[22]  S. Hell,et al.  STED microscopy detects and quantifies liquid phase separation in lipid membranes using a new far-red emitting fluorescent phosphoglycerolipid analogue. , 2013, Faraday discussions.

[23]  H. Hess,et al.  Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM. , 2014, Methods in cell biology.

[24]  J. Sethna,et al.  Critical Casimir forces in cellular membranes. , 2012, Physical review letters.

[25]  Elliot L Elson,et al.  Phase separation in biological membranes: integration of theory and experiment. , 2010, Annual review of biophysics.

[26]  Coordinate-based co-localization-mediated analysis of arrestin clustering upon stimulation of the C–C chemokine receptor 5 with RANTES/CCL5 analogues , 2014, Histochemistry and Cell Biology.

[27]  Masahiko Watanabe,et al.  Cell-specific STORM superresolution imaging reveals nanoscale organization of cannabinoid signaling , 2014, Nature Neuroscience.

[28]  P. Yeagle Cholesterol and the cell membrane. , 1985, Biochimica et biophysica acta.

[29]  Wonhwa Cho,et al.  Membrane-protein interactions in cell signaling and membrane trafficking. , 2005, Annual review of biophysics and biomolecular structure.

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

[31]  N. Thompson,et al.  Total internal reflection fluorescence. , 1984, Annual review of biophysics and bioengineering.

[32]  Kai Simons,et al.  Revitalizing membrane rafts: new tools and insights , 2010, Nature Reviews Molecular Cell Biology.

[33]  Thomas Huser,et al.  Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations , 2015, Scientific Reports.

[34]  Travis J Gould,et al.  Actin mediates the nanoscale membrane organization of the clustered membrane protein influenza hemagglutinin. , 2013, Biophysical journal.

[35]  S J Singer,et al.  The molecular organization of membranes. , 1974, Annual review of biochemistry.

[36]  Bridget S. Wilson,et al.  Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton , 2006, Proceedings of the National Academy of Sciences.

[37]  I. Huhtaniemi,et al.  Single Molecule Analysis of Functionally Asymmetric G Protein-coupled Receptor (GPCR) Oligomers Reveals Diverse Spatial and Structural Assemblies*♦ , 2014, The Journal of Biological Chemistry.

[38]  Mark M Davis,et al.  TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation , 2010, Nature Immunology.

[39]  Joel M. Harris,et al.  Super‐Resolution Imaging and Quantitative Analysis of Membrane Protein/Lipid Raft Clustering Mediated by Cell‐Surface Self‐Assembly of Hybrid Nanoconjugates , 2015, Chembiochem : a European journal of chemical biology.

[40]  Gleb Shtengel,et al.  Correlative super-resolution fluorescence and metal replica transmission electron microscopy , 2014, Nature Methods.

[41]  S W Hell,et al.  STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells. , 2011, Biophysical journal.

[42]  P. Lappalainen,et al.  Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides. , 2010, Physiological reviews.

[43]  R. Tsien,et al.  Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein , 2004, Nature Biotechnology.

[44]  K. Lidke,et al.  Actin restricts FcɛRI diffusion and facilitates antigen-induced receptor immobilization , 2008, Nature Cell Biology.

[45]  C. Riener,et al.  Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin. , 2000, Bioconjugate chemistry.

[46]  Michel Bouvier,et al.  Oligomerization of G-protein-coupled transmitter receptors , 2001, Nature Reviews Neuroscience.

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

[48]  Ilan Davis,et al.  Super-resolution imaging of remodeled synaptic actin reveals different synergies between NK cell receptors and integrins. , 2012, Blood.

[49]  Till Bretschneider,et al.  Subsecond reorganization of the actin network in cell motility and chemotaxis. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[50]  A. Kusumi,et al.  Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. , 1993, Biophysical journal.

[51]  Suliana Manley,et al.  Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor. , 2011, Immunity.

[52]  K. B. Oldham A Gouy–Chapman–Stern model of the double layer at a (metal)/(ionic liquid) interface , 2008 .

[53]  D. Axelrod Cell-substrate contacts illuminated by total internal reflection fluorescence , 1981, The Journal of cell biology.

[54]  M. A. Surma,et al.  Polyunsaturated Lipids Regulate Membrane Domain Stability by Tuning Membrane Order. , 2016, Biophysical journal.

[55]  Diana Murray,et al.  PIP(2) and proteins: interactions, organization, and information flow. , 2002, Annual review of biophysics and biomolecular structure.

[56]  A. Shevchenko,et al.  Lipidomics: coming to grips with lipid diversity , 2010, Nature Reviews Molecular Cell Biology.

[57]  Sudeep Banjade,et al.  Phase transitions of multivalent proteins can promote clustering of membrane receptors , 2014, eLife.

[58]  D. Richards,et al.  Segregation of PIP2 and PIP3 into distinct nanoscale regions within the plasma membrane , 2012, Biology Open.

[59]  Samuel T. Hess,et al.  Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo , 2015, PloS one.

[60]  Roland Eils,et al.  One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy , 2015, Scientific Reports.

[61]  Sean Quirin,et al.  Optimal 3D single-molecule localization for superresolution microscopy with aberrations and engineered point spread functions , 2011, Proceedings of the National Academy of Sciences.

[62]  A Radenovic,et al.  Challenges in quantitative single molecule localization microscopy , 2014, FEBS letters.

[63]  P. Annibale,et al.  Cell Type-specific β2-Adrenergic Receptor Clusters Identified Using Photoactivated Localization Microscopy Are Not Lipid Raft Related, but Depend on Actin Cytoskeleton Integrity* , 2012, The Journal of Biological Chemistry.

[64]  Yongdeng Zhang,et al.  Nanoscale Landscape of Phosphoinositides Revealed by Specific Pleckstrin Homology (PH) Domains Using Single-molecule Superresolution Imaging in the Plasma Membrane* , 2015, The Journal of Biological Chemistry.

[65]  Krishnan Raghunathan,et al.  Glycolipid Crosslinking Is Required for Cholera Toxin to Partition Into and Stabilize Ordered Domains. , 2016, Biophysical journal.

[66]  J. Seddon,et al.  Structure of the inverted hexagonal (HII) phase, and non-lamellar phase transitions of lipids. , 1990, Biochimica et biophysica acta.

[67]  Christer S. Ejsing,et al.  Homeoviscous Adaptation and the Regulation of Membrane Lipids. , 2016, Journal of molecular biology.

[68]  G. Meer,et al.  Membrane lipids: where they are and how they behave , 2008, Nature Reviews Molecular Cell Biology.

[69]  M. Sheetz,et al.  Nanometer-scale measurements using video light microscopy. , 1988, Cell motility and the cytoskeleton.

[70]  M. Bruchez,et al.  Fluorogen-Activating Proteins Provide Tunable Labeling Densities for Tracking FcεRI Independent of IgE , 2014, ACS chemical biology.

[71]  Endre Kiss,et al.  Imaging of Mobile Long-lived Nanoplatforms in the Live Cell Plasma Membrane* , 2010, The Journal of Biological Chemistry.

[72]  Carla Coltharp,et al.  Accurate Construction of Photoactivated Localization Microscopy (PALM) Images for Quantitative Measurements , 2012, PloS one.

[73]  Z. Gong,et al.  Super Resolution Microscopy Reveals that Caveolin-1 Is Required for Spatial Organization of CRFB1 and Subsequent Antiviral Signaling in Zebrafish , 2013, PloS one.

[74]  D. Jackson,et al.  Critical importance of appropriate fixation conditions for faithful imaging of receptor microclusters , 2016, Biology Open.

[75]  J. Moss,et al.  Cyclic AMP-independent effects of cholera toxin on B cell activation. II. Binding of ganglioside GM1 induces B cell activation. , 1992, Journal of immunology.

[76]  Kathryn L. Schornberg,et al.  Structures and Mechanisms of Viral Membrane Fusion Proteins: Multiple Variations on a Common Theme , 2008 .

[77]  C. Kelly,et al.  Revealing Nanoscale Membrane Curvature with Polarized Localization Microscopy , 2015 .

[78]  Katharina Gaus,et al.  Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination , 2016, Proceedings of the National Academy of Sciences.

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

[80]  Hongbin Ji,et al.  Mechanistic insights into EGFR membrane clustering revealed by super-resolution imaging. , 2015, Nanoscale.

[81]  Mike Heilemann,et al.  Super-resolution Imaging Reveals the Internal Architecture of Nano-sized Syntaxin Clusters* , 2012, The Journal of Biological Chemistry.

[82]  Colin K. Choi,et al.  Stoichiometry of molecular complexes at adhesions in living cells , 2009, Proceedings of the National Academy of Sciences.

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

[84]  Valerie C. Coffman,et al.  Counting protein molecules using quantitative fluorescence microscopy. , 2012, Trends in biochemical sciences.

[85]  S. Hell,et al.  A lipid bound actin meshwork organizes liquid phase separation in model membranes , 2014, eLife.

[86]  D. Lingwood,et al.  Palmitoylation regulates raft affinity for the majority of integral raft proteins , 2010, Proceedings of the National Academy of Sciences.

[87]  E J Luna,et al.  Cytoskeleton--plasma membrane interactions. , 1992, Science.

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

[89]  Hans-Peter Kriegel,et al.  A Density-Based Algorithm for Discovering Clusters in Large Spatial Databases with Noise , 1996, KDD.

[90]  J. Spudich,et al.  Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[91]  K. Kremer,et al.  Aggregation and vesiculation of membrane proteins by curvature-mediated interactions , 2007, Nature.

[92]  Matthew B Stone,et al.  Far-red organic fluorophores contain a fluorescent impurity. , 2014, Chemphyschem : a European journal of chemical physics and physical chemistry.

[93]  R. McElhaney,et al.  Membrane lipid phase transitions and phase organization studied by Fourier transform infrared spectroscopy. , 2013, Biochimica et biophysica acta.

[94]  S. Rasmussen,et al.  The structure and function of G-protein-coupled receptors , 2009, Nature.

[95]  J. Martial,et al.  Crystal structure of cholera toxin B‐pentamer bound to receptor GM1 pentasaccharide , 1994, Protein science : a publication of the Protein Society.

[96]  J. Hörber,et al.  Sphingolipid–Cholesterol Rafts Diffuse as Small Entities in the Plasma Membrane of Mammalian Cells , 2000, The Journal of cell biology.

[97]  Andrei Faraon,et al.  Removing Orientation-Induced Localization Biases in Single-Molecule Microscopy Using a Broadband Metasurface Mask , 2016, Nature Photonics.

[98]  Lucien E. Weiss,et al.  Precise Three-Dimensional Scan-Free Multiple-Particle Tracking over Large Axial Ranges with Tetrapod Point Spread Functions , 2015, Nano letters.

[99]  L. Tamm,et al.  Clustering of syntaxin-1A in model membranes is modulated by phosphatidylinositol 4,5-bisphosphate and cholesterol. , 2009, Biochemistry.

[100]  A. Gavin,et al.  A Conserved Circular Network of Coregulated Lipids Modulates Innate Immune Responses , 2015, Cell.

[101]  D. Kovar,et al.  JCB_201502062 1..9 , 2015 .

[102]  Martin Caffrey,et al.  A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes , 2015, Acta crystallographica. Section F, Structural biology communications.

[103]  M. Lemmon,et al.  Membrane recognition by phospholipid-binding domains , 2008, Nature Reviews Molecular Cell Biology.

[104]  B. Baird,et al.  Functional nanoscale coupling of Lyn kinase with IgE-FcεRI is restricted by the actin cytoskeleton in early antigen-stimulated signaling , 2016, Molecular biology of the cell.

[105]  S. Hell,et al.  Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane – a minimally invasive investigation by STED-FCS , 2015, Scientific Reports.

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

[107]  A. McClatchey,et al.  Ezrin Tunes the Magnitude of Humoral Immunity , 2013, The Journal of Immunology.

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

[109]  H. Heerklotz Triton promotes domain formation in lipid raft mixtures. , 2002, Biophysical journal.

[110]  Patrick W. Oakes,et al.  Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton , 2016, The Journal of cell biology.

[111]  Prabuddha Sengupta,et al.  The nanoscale spatial organization of B-cell receptors on immunoglobulin M– and G–expressing human B-cells , 2017, Molecular biology of the cell.

[112]  J. Seelig,et al.  The sensitivity of lipid domains to small perturbations demonstrated by the effect of Triton. , 2003, Journal of molecular biology.

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

[114]  J. J. Macklin,et al.  A general method to improve fluorophores for live-cell and single-molecule microscopy , 2014, Nature Methods.

[115]  B. Baird,et al.  Distinct stages of stimulated FcεRI receptor clustering and immobilization are identified through superresolution imaging. , 2013, Biophysical journal.

[116]  Steve Pressé,et al.  Stochastic approach to the molecular counting problem in superresolution microscopy , 2014, Proceedings of the National Academy of Sciences.

[117]  R Nuydens,et al.  Lateral diffusion and retrograde movements of individual cell surface components on single motile cells observed with Nanovid microscopy , 1991, The Journal of cell biology.

[118]  E. Ikonen,et al.  Functional rafts in cell membranes , 1997, Nature.

[119]  K. Jacobson,et al.  Super-resolution imaging of C-type lectin and influenza hemagglutinin nanodomains on plasma membranes using blink microscopy. , 2012, Biophysical journal.

[120]  S. Veatch,et al.  Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes , 2017, eLife.

[121]  S. Linder,et al.  Podosomes at a glance , 2005, Journal of Cell Science.

[122]  T. Steck THE ORGANIZATION OF PROTEINS IN THE HUMAN RED BLOOD CELL MEMBRANE , 1974, The Journal of cell biology.

[123]  Hayder Amin,et al.  Membrane protein sequestering by ionic protein-lipid interactions , 2011, Nature.

[124]  Ying S Hu,et al.  Superresolution imaging reveals nanometer- and micrometer-scale spatial distributions of T-cell receptors in lymph nodes , 2016, Proceedings of the National Academy of Sciences.

[125]  Astrid Magenau,et al.  PALM imaging and cluster analysis of protein heterogeneity at the cell surface , 2010, Journal of biophotonics.

[126]  Andreas Bruckbauer,et al.  The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling. , 2013, Immunity.

[127]  J. Hurley,et al.  Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly , 2015, Proceedings of the National Academy of Sciences.

[128]  H. Metzger,et al.  Transmembrane signaling: the joy of aggregation. , 1992, Journal of immunology.

[129]  M. Sheetz,et al.  Tracking kinesin-driven movements with nanometre-scale precision , 1988, Nature.

[130]  Michael W. Davidson,et al.  A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells , 2014, The Journal of cell biology.

[131]  K. Jacobson,et al.  New insights into membrane dynamics from the analysis of cell surface interactions by physical methods. , 1995, Current opinion in cell biology.

[132]  Jürgen Wolfrum,et al.  Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan. , 2003, Bioconjugate chemistry.

[133]  J. Bewersdorf,et al.  Three dimensional single molecule localization using a phase retrieved pupil function. , 2013, Optics express.

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

[135]  M. Sheetz,et al.  Continuous membrane-cytoskeleton adhesion requires continuous accommodation to lipid and cytoskeleton dynamics. , 2006, Annual review of biophysics and biomolecular structure.

[136]  J. Bertin,et al.  The immune receptor NOD1 and kinase RIP2 interact with bacterial peptidoglycan on early endosomes to promote autophagy and inflammatory signaling. , 2014, Cell host & microbe.

[137]  J. Petrich,et al.  Tryptophan and ATTO 590: mutual fluorescence quenching and exciplex formation. , 2014, The journal of physical chemistry. B.

[138]  Steven F. Lee,et al.  Improved super-resolution microscopy with oxazine fluorophores in heavy water. , 2013, Angewandte Chemie.

[139]  Daniel Wüstner,et al.  Analysis of cholesterol trafficking with fluorescent probes. , 2012, Methods in cell biology.

[140]  G. Bachand,et al.  Engineering Lipid Structure for Recognition of the Liquid Ordered Membrane Phase. , 2016, Langmuir : the ACS journal of surfaces and colloids.

[141]  T. Jovanović-Talisman,et al.  Nanoscale Effects of Ethanol and Naltrexone on Protein Organization in the Plasma Membrane Studied by Photoactivated Localization Microscopy (PALM) , 2014, PloS one.

[142]  T. Südhof,et al.  Membrane Fusion: Grappling with SNARE and SM Proteins , 2009, Science.

[143]  Christian Eggeling,et al.  Super-Resolved Traction Force Microscopy (STFM) , 2016, Nano letters.

[144]  T. Harder,et al.  Clusters of glycolipid and glycosylphosphatidylinositol‐anchored proteins in lymphoid cells : accumulation of actin regulated by local tyrosine phosphorylation , 1999, European journal of immunology.

[145]  M. Resh,et al.  Trafficking and signaling by fatty-acylated and prenylated proteins , 2006, Nature chemical biology.

[146]  Christian Eggeling,et al.  Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells , 2014, Nature Communications.

[147]  A. Kenworthy,et al.  Tracking microdomain dynamics in cell membranes. , 2009, Biochimica et biophysica acta.

[148]  P. Janmey,et al.  Divalent cation-induced cluster formation by polyphosphoinositides in model membranes. , 2012, Journal of the American Chemical Society.

[149]  J. Dodge,et al.  The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. , 1963, Archives of biochemistry and biophysics.

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

[151]  R. Parton,et al.  The multiple faces of caveolae , 2007, Nature Reviews Molecular Cell Biology.

[152]  Jeffrey R Moffitt,et al.  Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging , 2014, Proceedings of the National Academy of Sciences.

[153]  Yongdeng Zhang,et al.  Rational design of true monomeric and bright photoactivatable fluorescent proteins , 2012, Nature Methods.

[154]  Ji Yi,et al.  Super-resolution two-photon microscopy via scanning patterned illumination. , 2015, Physical review. E, Statistical, nonlinear, and soft matter physics.

[155]  Christian Eggeling,et al.  rsEGFP2 enables fast RESOLFT nanoscopy of living cells , 2012, eLife.

[156]  G. Patterson,et al.  Axial superresolution via multiangle TIRF microscopy with sequential imaging and photobleaching , 2016, Proceedings of the National Academy of Sciences.

[157]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[158]  Astrid Magenau,et al.  Sub-resolution lipid domains exist in the plasma membrane and regulate protein diffusion and distribution , 2012, Nature Communications.

[159]  A. Diaspro,et al.  Correction: Light-Sheet Confined Super-Resolution Using Two-Photon Photoactivation , 2013, PLoS ONE.

[160]  S. Hell,et al.  Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains , 2015, Nature Communications.

[161]  E. Sackmann,et al.  Adhesion-induced domain formation by interplay of long-range repulsion and short-range attraction force: a model membrane study. , 1997, Biophysical journal.

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

[163]  L. Hunyady,et al.  Dimerization and oligomerization of G-protein-coupled receptors: debated structures with established and emerging functions. , 2008, The Journal of endocrinology.

[164]  U. Endesfelder,et al.  Quantitative morphological analysis of arrestin2 clustering upon G protein-coupled receptor stimulation by super-resolution microscopy. , 2013, Journal of structural biology.

[165]  P. Mattila,et al.  Dynamics of the actin cytoskeleton mediates receptor cross talk: An emerging concept in tuning receptor signaling , 2016, The Journal of cell biology.

[166]  Benoît Roux,et al.  Binding specificity of SH2 domains: Insight from free energy simulations , 2009, Proteins.

[167]  R. Edwards,et al.  Localized topological changes of the plasma membrane upon exocytosis visualized by polarized TIRFM , 2010, The Journal of cell biology.

[168]  M. Yuseff,et al.  How B cells capture, process and present antigens: a crucial role for cell polarity , 2013, Nature Reviews Immunology.

[169]  Gary G. Borisy,et al.  Mechanism of filopodia initiation by reorganization of a dendritic network , 2003, The Journal of cell biology.

[170]  M. Brameshuber,et al.  GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane , 2015, Nature Communications.

[171]  O. Ronneberger,et al.  B cell antigen receptors of the IgM and IgD classes are clustered in different protein islands that are altered during B cell activation , 2015, Science Signaling.

[172]  Joshua C Vaughan,et al.  Twinkle, twinkle little star: Photoswitchable fluorophores for super‐resolution imaging , 2014, FEBS letters.

[173]  G. Wadhams,et al.  Stoichiometry and turnover in single, functioning membrane protein complexes , 2006, Nature.

[174]  Stefan W. Hell,et al.  CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells , 2015, Scientific Reports.

[175]  Benjamin B. Machta,et al.  Correlation Functions Quantify Super-Resolution Images and Estimate Apparent Clustering Due to Over-Counting , 2011, PloS one.

[176]  Markus Sauer,et al.  Localization microscopy coming of age: from concepts to biological impact , 2013, Journal of Cell Science.

[177]  U. Endesfelder,et al.  Multiscale spatial organization of RNA polymerase in Escherichia coli. , 2013, Biophysical journal.

[178]  Sarah L Veatch,et al.  Seeing spots: complex phase behavior in simple membranes. , 2005, Biochimica et biophysica acta.

[179]  Lipid polymorphism and the functional roles of lipids in biological membranes. , 1979 .

[180]  Roger Y. Tsien,et al.  Improved green fluorescence , 1995, Nature.

[181]  Brian P. Mehl,et al.  Bright photoactivatable fluorophores for single-molecule imaging , 2016 .

[182]  Michael W. Davidson,et al.  mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities , 2012, PloS one.

[183]  M. Neil,et al.  Remodelling of Cortical Actin Where Lytic Granules Dock at Natural Killer Cell Immune Synapses Revealed by Super-Resolution Microscopy , 2011, PLoS biology.

[184]  Akihiro Kusumi,et al.  Single-molecule imaging revealed dynamic GPCR dimerization. , 2014, Current opinion in cell biology.

[185]  Conditions for extreme sensitivity of protein diffusion in membranes to cell environments , 2006, Proceedings of the National Academy of Sciences.

[186]  S. Hell,et al.  Direct observation of the nanoscale dynamics of membrane lipids in a living cell , 2009, Nature.

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

[188]  Sjoerd Stallinga,et al.  Measuring image resolution in optical nanoscopy , 2013, Nature Methods.

[189]  Mark Bates,et al.  Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes , 2007, Science.

[190]  D. Golan,et al.  Use of a fluorescent cholesterol derivative to measure lateral mobility of cholesterol in membranes. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[191]  Wei Zhang,et al.  Correction of depth-dependent aberrations in 3D single-molecule localization and super-resolution microscopy. , 2014, Optics letters.

[192]  S. Gruner,et al.  Lipid polymorphism: the molecular basis of nonbilayer phases. , 1985, Annual review of biophysics and biophysical chemistry.

[193]  James A J Fitzpatrick,et al.  Fluorogen-activating single-chain antibodies for imaging cell surface proteins , 2008, Nature Biotechnology.

[194]  Watt W. Webb,et al.  Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells , 2005, The Journal of cell biology.

[195]  Wilhelm Friedrich,et al.  Lymphocyte microvilli are dynamic, actin-dependent structures that do not require Wiskott-Aldrich syndrome protein (WASp) for their morphology , 2004 .

[196]  W. Webb,et al.  Constrained diffusion or immobile fraction on cell surfaces: a new interpretation. , 1996, Biophysical journal.

[197]  L. Johnston,et al.  Imaging nanometer domains of β-adrenergic receptor complexes on the surface of cardiac myocytes , 2005, Nature chemical biology.

[198]  Akihiro Kusumi,et al.  Hierarchical organization of the plasma membrane: Investigations by single‐molecule tracking vs. fluorescence correlation spectroscopy , 2010, FEBS letters.

[199]  Laurent Cognet,et al.  Identification and super-resolution imaging of ligand-activated receptor dimers in live cells , 2013, Scientific Reports.

[200]  O. Ohara,et al.  Single molecular assay of individual ATP turnover by a myosin‐GFP fusion protein expressed in vitro , 1997, FEBS letters.

[201]  S. Hell,et al.  Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell , 2008, Proceedings of the National Academy of Sciences.

[202]  Astrid Magenau,et al.  Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events , 2011, Nature Immunology.

[203]  Akihiro Kusumi,et al.  Tracking single molecules at work in living cells. , 2014, Nature chemical biology.

[204]  T. Huber,et al.  Labeling and Single-Molecule Methods To Monitor G Protein-Coupled Receptor Dynamics. , 2017, Chemical reviews.

[205]  H. Sandermann High free energy of lipid/protein interaction in biological membranes , 2002, FEBS letters.

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

[207]  R. Mitchell,et al.  A Role for Lipid Rafts in B Cell Antigen Receptor Signaling and Antigen Targeting , 1999, The Journal of experimental medicine.

[208]  J. Sethna,et al.  Minimal model of plasma membrane heterogeneity requires coupling cortical actin to criticality. , 2010, Biophysical journal.

[209]  W. Webb,et al.  Fluorescent low density lipoprotein for observation of dynamics of individual receptor complexes on cultured human fibroblasts , 1981, The Journal of cell biology.

[210]  Frederick A. Heberle,et al.  Phase separation in lipid membranes. , 2011, Cold Spring Harbor perspectives in biology.

[211]  Enrico Gratton,et al.  Fast spatiotemporal correlation spectroscopy to determine protein lateral diffusion laws in live cell membranes , 2013, Proceedings of the National Academy of Sciences.

[212]  A. Kenworthy Have we become overly reliant on lipid rafts? , 2008, EMBO reports.

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

[214]  S. Hell,et al.  Reorganization of Lipid Diffusion by Myelin Basic Protein as Revealed by STED Nanoscopy , 2016, Biophysical journal.

[215]  R Nuydens,et al.  Probing microtubule-dependent intracellular motility with nanometre particle video ultramicroscopy (nanovid ultramicroscopy). , 1985, Cytobios.

[216]  M. Bathe,et al.  Bayesian total internal reflection fluorescence correlation spectroscopy reveals hIAPP-induced plasma membrane domain organization in live cells. , 2014, Biophysical journal.

[217]  Ahmed Elmokadem,et al.  Optimal Drift Correction for Superresolution Localization Microscopy with Bayesian Inference. , 2015, Biophysical journal.

[218]  R. A. Cooper Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells. , 1978, Journal of supramolecular structure.

[219]  Xavier Deupi,et al.  Conformational complexity of G-protein-coupled receptors. , 2007, Trends in pharmacological sciences.

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

[221]  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.

[222]  Takeharu Nagai,et al.  Shift anticipated in DNA microarray market , 2002, Nature Biotechnology.

[223]  K. Nagashima,et al.  Roles Played by Capsid-Dependent Induction of Membrane Curvature and Gag-ESCRT Interactions in Tetherin Recruitment to HIV-1 Assembly Sites , 2013, Journal of Virology.

[224]  B. Tenchov On the reversibility of the phase transitions in lipid-water systems. , 1991, Chemistry and physics of lipids.

[225]  Ronald D. Vale,et al.  Imaging individual green fluorescent proteins , 1997, Nature.

[226]  I. Levental,et al.  The Continuing Mystery of Lipid Rafts. , 2016, Journal of molecular biology.

[227]  Stefan W Hell,et al.  STED nanoscopy reveals the ubiquity of subcortical cytoskeleton periodicity in living neurons. , 2015, Cell reports.

[228]  G. Hummer,et al.  Model-independent counting of molecules in single-molecule localization microscopy , 2016, Molecular biology of the cell.

[229]  Igor Orlov,et al.  ClusterViSu, a method for clustering of protein complexes by Voronoi tessellation in super-resolution microscopy , 2016, Scientific Reports.

[230]  A Kusumi,et al.  Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometer-level motion analysis , 1994, The Journal of cell biology.

[231]  B. Önfelt,et al.  Consequences of membrane topography , 2013, The FEBS journal.

[232]  S. Veatch,et al.  Oxygen depletion speeds and simplifies diffusion in HeLa cells. , 2014, Biophysical journal.

[233]  J. Thyberg,et al.  Microvilli structures on B lymphocytes: inducible functional domains? , 2004, International immunology.

[234]  Bhaswati Bhattacharya,et al.  Spatiotemporal regulation of chemical reactions by active cytoskeletal remodeling , 2011, Proceedings of the National Academy of Sciences.

[235]  Robert J. Lefkowitz,et al.  Transduction of Receptor Signals by ß-Arrestins , 2005, Science.

[236]  Samuel T. Hess,et al.  Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories , 2007, Proceedings of the National Academy of Sciences.

[237]  Aashish Manglik,et al.  Propagation of conformational changes during μ-opioid receptor activation , 2015, Nature.

[238]  Frederick A. Heberle,et al.  Crosslinking a lipid raft component triggers liquid ordered-liquid disordered phase separation in model plasma membranes. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[239]  Péter Várnai,et al.  Visualization of Phosphoinositides That Bind Pleckstrin Homology Domains: Calcium- and Agonist-induced Dynamic Changes and Relationship to Myo-[3H]inositol-labeled Phosphoinositide Pools , 1998, The Journal of cell biology.

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

[241]  J. Jouhet Importance of the hexagonal lipid phase in biological membrane organization , 2013, Front. Plant Sci..