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Single-molecule counting applied to the study of GPCR oligomerization

Published:August 03, 2022DOI:https://doi.org/10.1016/j.bpj.2022.07.034

      Abstract

      Single-molecule counting techniques enable a precise determination of the intracellular abundance and stoichiometry of proteins and macromolecular complexes. These details are often challenging to quantitatively assess yet are essential for our understanding of cellular function. Consider G-protein-coupled receptors—an expansive class of transmembrane signaling proteins that participate in many vital physiological functions making them a popular target for drug development. While early evidence for the role of oligomerization in receptor signaling came from ensemble biochemical and biophysical assays, innovations in single-molecule measurements are now driving a paradigm shift in our understanding of its relevance. Here, we review recent developments in single-molecule counting with a focus on photobleaching step counting and the emerging technique of quantitative single-molecule localization microscopy—with a particular emphasis on the potential for these techniques to advance our understanding of the role of oligomerization in G-protein-coupled receptor signaling.
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      References

        • Ishikawa K.
        Multilayered regulation of proteome stoichiometry.
        Curr. Genet. 2021; 67: 883-890
        • Marsh J.A.
        • Teichmann S.A.
        Structure, dynamics, assembly, and evolution of protein complexes.
        Annu. Rev. Biochem. 2015; 84: 551-575
        • Clark N.M.
        • Fisher A.P.
        • Sozzani R.
        • et al.
        Protein complex stoichiometry and expression dynamics of transcription factors modulate stem cell division.
        Proc. Natl. Acad. Sci. USA. 2020; 117: 15332-15342
        • Brandenberg O.F.
        • Magnus C.
        • Trkola A.
        • et al.
        Predicting HIV-1 transmission and antibody neutralization efficacy in vivo from stoichiometric parameters.
        PLoS Pathog. 2017; 13: e1006313
        • Fredriksson R.
        • Lagerström M.C.
        • Schiöth H.B.
        • et al.
        The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints.
        Mol. Pharmacol. 2003; 63: 1256-1272
        • Katritch V.
        • Cherezov V.
        • Stevens R.C.
        Diversity and modularity of G protein-coupled receptor structures.
        Trends Pharmacol. Sci. 2012; 33: 17-27
        • Sriram K.
        • Insel P.A.
        GPCRs as targets for approved drugs: how many targets and how many drugs?.
        Mol. Pharmacol. 2018; https://doi.org/10.1124/mol.117.111062
        • Juette M.F.
        • Terry D.S.
        • Blanchard S.C.
        • et al.
        Single-molecule imaging of non-equilibrium molecular ensembles on the millisecond timescale.
        Nat. Methods. 2016; 13: 341-344
        • Tian H.
        • Fürstenberg A.
        • Huber T.
        Labeling and single-molecule methods to monitor G protein-coupled receptor dynamics.
        Chem. Rev. 2017; 117: 186-245
        • Felce J.H.
        • Latty S.L.
        • Davis S.J.
        • et al.
        Receptor quaternary organization explains G protein-coupled receptor family structure.
        Cell Rep. 2017; 20: 2654-2665
        • Rosenbaum D.M.
        • Rasmussen S.G.F.
        • Kobilka B.K.
        The structure and function of G-protein-coupled receptors.
        Nature. 2009; 459: 356-363
        • Manglik A.
        • Kruse A.C.
        Structural basis for G protein-coupled receptor activation.
        Biochemistry. 2017; 56: 5628-5634
        • Ferré S.
        • Casadó V.
        • Guitart X.
        • et al.
        G protein–coupled receptor oligomerization revisited: functional and pharmacological perspectives.
        Pharmacol. Rev. 2014; 66: 413-434
        • Milligan G.
        • Ward R.J.
        • Marsango S.
        GPCR homo-oligomerization.
        Curr. Opin. Cell Biol. 2019; 57: 40-47
        • Sleno R.
        • Hébert T.E.
        Shaky ground - the nature of metastable GPCR signalling complexes.
        Neuropharmacology. 2019; 152: 4-14
        • von Diezmann L.
        • Shechtman Y.
        • Moerner W.E.
        Three-dimensional localization of single molecules for super-resolution imaging and single-particle tracking.
        Chem. Rev. 2017; 117: 7244-7275
        • Feng X.A.
        • Poyton M.F.
        • Ha T.
        Multicolor single-molecule FRET for DNA and RNA processes.
        Curr. Opin. Struct. Biol. 2021; 70: 26-33
        • Ghosh A.
        • Enderlein J.
        Advanced fluorescence correlation spectroscopy for studying biomolecular conformation.
        Curr. Opin. Struct. Biol. 2021; 70: 123-131
        • Gong W.
        • Das P.
        • Yang Z.
        • et al.
        Redefining the photo-stability of common fluorophores with triplet state quenchers: mechanistic insights and recent updates.
        Chem. Commun. 2019; 55: 8695-8704
        • Gordon M.P.
        • Ha T.
        • Selvin P.R.
        Single-molecule high-resolution imaging with photobleaching.
        Proc. Natl. Acad. Sci. USA. 2004; 101: 6462-6465
        • Engel B.D.
        • Ludington W.B.
        • Marshall W.F.
        Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model.
        J. Cell Biol. 2009; 187: 81-89
        • Ulbrich M.H.
        • Isacoff E.Y.
        Subunit counting in membrane-bound proteins.
        Nat. Methods. 2007; 4: 319-321
        • Leake M.C.
        • Chandler J.H.
        • Armitage J.P.
        • et al.
        Stoichiometry and turnover in single, functioning membrane protein complexes.
        Nature. 2006; 443: 355-358
        • Das S.K.
        • Darshi M.
        • Bayley H.
        • et al.
        Membrane protein stoichiometry determined from the step-wise photobleaching of dye-labelled subunits.
        Chembiochem. 2007; 8: 994-999
        • Gallistel C.R.
        • Fairhurst S.
        • Balsam P.
        The learning curve: implications of a quantitative analysis.
        Proc. Natl. Acad. Sci. USA. 2004; 101: 13124-13131
        • Shivnaraine R.V.
        • Fernandes D.D.
        • Gradinaru C.C.
        • et al.
        Single-molecule analysis of the supramolecular organization of the M2 muscarinic receptor and the Gαi1 protein.
        J. Am. Chem. Soc. 2016; 138: 11583-11598
        • Tsekouras K.
        • Custer T.C.
        • Pressé S.
        • et al.
        A novel method to accurately locate and count large numbers of steps by photobleaching.
        Mol. Biol. Cell. 2016; 27: 3601-3615
        • Hummert J.
        • Yserentant K.
        • Herten D.-P.
        • et al.
        Photobleaching step analysis for robust determination of protein complex stoichiometries.
        Mol. Biol. Cell. 2021; 32: ar35
        • Li H.
        • Yang H.
        Statistical learning of discrete states in time series.
        J. Phys. Chem. B. 2019; 123: 689-701
        • Garry J.
        • Li Y.
        • Rutenberg A.D.
        • et al.
        Bayesian counting of photobleaching steps with physical priors.
        J. Chem. Phys. 2020; 152: 024110
        • Heilemann M.
        • Van De Linde S.
        • Sauer M.
        • et al.
        Super-resolution imaging with small organic fluorophores.
        Angew. Chem. Int. Ed. Engl. 2009; 48: 6903-6908
        • Heilemann M.
        • van de Linde S.
        • Sauer M.
        • et al.
        Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes.
        Angew. Chem. Int. Ed. Engl. 2008; 47: 6172-6176
        • Rust M.J.
        • Bates M.
        • Zhuang X.
        Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).
        Nat. Methods. 2006; 3: 793-795
        • Betzig E.
        • Patterson G.H.
        • Hess H.F.
        • et al.
        Imaging intracellular fluorescent proteins at nanometer resolution.
        Science. 2006; 313: 1642-1645
        • Nan X.
        • Collisson E.A.
        • Chu S.
        • et al.
        Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling.
        Proc. Natl. Acad. Sci. USA. 2013; 110: 18519-18524
        • Jungmann R.
        • Steinhauer C.
        • Simmel F.C.
        • et al.
        Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami.
        Nano Lett. 2010; 10: 4756-4761
        • Schnitzbauer J.
        • Strauss M.T.
        • Jungmann R.
        • et al.
        Super-resolution microscopy with DNA-PAINT.
        Nat. Protoc. 2017; 12: 1198-1228
        • Balzarotti F.
        • Eilers Y.
        • Hell S.W.
        • et al.
        Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.
        Science. 2017; 355: 606-612
        • Schmidt R.
        • Weihs T.
        • Hell S.W.
        • et al.
        MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope.
        Nat. Commun. 2021; 12: 1478
        • Gu L.
        • Li Y.
        • Ji W.
        • et al.
        Molecular resolution imaging by repetitive optical selective exposure.
        Nat. Methods. 2019; 16: 1114-1118
        • Reymond L.
        • Ziegler J.
        • Wieser S.
        • et al.
        SIMPLE: structured illumination based point localization estimator with enhanced precision.
        Opt Express. 2019; 27: 24578-24590
        • Shivanandan A.
        • Deschout H.
        • Radenovic A.
        • et al.
        Challenges in quantitative single molecule localization microscopy.
        FEBS Lett. 2014; 588: 3595-3602
        • Baddeley D.
        • Bewersdorf J.
        Biological insight from super-resolution microscopy: what we can learn from localization-based images.
        Annu. Rev. Biochem. 2018; 87: 965-989
        • Jung S.-R.
        • Fujimoto B.S.
        • Chiu D.T.
        Quantitative microscopy based on single-molecule fluorescence.
        Curr. Opin. Chem. Biol. 2017; 39: 64-73
        • Jungmann R.
        • Avendaño M.S.
        • Yin P.
        • et al.
        Quantitative super-resolution imaging with qPAINT.
        Nat. Methods. 2016; 13: 439-442
        • Annibale P.
        • Vanni S.
        • Radenovic A.
        • et al.
        Quantitative photo activated localization microscopy: unraveling the effects of photoblinking.
        PLoS One. 2011; 6: e22678
        • Deschout H.
        • Shivanandan A.
        • Radenovic A.
        • et al.
        Progress in quantitative single-molecule localization microscopy.
        Histochem. Cell Biol. 2014; 142: 5-17
        • Durisic N.
        • Cuervo L.L.
        • Lakadamyali M.
        Quantitative super-resolution microscopy: pitfalls and strategies for image analysis.
        Curr. Opin. Chem. Biol. 2014; 20: 22-28
        • Annibale P.
        • Vanni S.
        • Radenovic A.
        • et al.
        Identification of clustering artifacts in photoactivated localization microscopy.
        Nat. Methods. 2011; 8: 527-528
        • Rossboth B.
        • Arnold A.M.
        • Schütz G.J.
        • et al.
        TCRs are randomly distributed on the plasma membrane of resting antigen-experienced T cells.
        Nat. Immunol. 2018; 19: 821-827
        • Sengupta P.
        • Jovanovic-Talisman T.
        • Lippincott-Schwartz J.
        • et al.
        Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis.
        Nat. Methods. 2011; 8: 969-975
        • Sengupta P.
        • Jovanovic-Talisman T.
        • Lippincott-Schwartz J.
        Quantifying spatial organization in point-localization superresolution images using pair correlation analysis.
        Nat. Protoc. 2013; 8: 345-354
        • Bohrer C.H.
        • Yang X.
        • Xiao J.
        • et al.
        A pairwise distance distribution correction (DDC) algorithm to eliminate blinking-caused artifacts in SMLM.
        Nat. Methods. 2021; 18: 669-677
        • Lee S.-H.
        • Shin J.Y.
        • Bustamante C.
        • et al.
        Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM).
        Proc. Natl. Acad. Sci. USA. 2012; 109: 17436-17441
        • Puchner E.M.
        • Walter J.M.
        • Lim W.A.
        • et al.
        Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory.
        Proc. Natl. Acad. Sci. USA. 2013; 110: 16015-16020
        • Coltharp C.
        • Kessler R.P.
        • Xiao J.
        Accurate construction of photoactivated localization microscopy (PALM) images for quantitative measurements.
        PLoS One. 2012; 7: e51725
        • Lillemeier B.F.
        • Mörtelmaier M.A.
        • Davis M.M.
        • et al.
        TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation.
        Nat. Immunol. 2010; 11: 90-96
        • Buss J.
        • Coltharp C.
        • Xiao J.
        • et al.
        In vivo organization of the FtsZ-ring by ZapA and ZapB revealed by quantitative super-resolution microscopy.
        Mol. Microbiol. 2013; 89: 1099-1120
        • Patel L.
        • Gustafsson N.
        • Cohen E.
        • et al.
        A hidden Markov model approach to characterizing the photo-switching behavior of fluorophores.
        Ann. Appl. Stat. 2019; 13: 1397-1429
        • Rollins G.C.
        • Shin J.Y.
        • Pressé S.
        • et al.
        Stochastic approach to the molecular counting problem in superresolution microscopy.
        Proc. Natl. Acad. Sci. USA. 2015; 112: E110-E118
        • Nieuwenhuizen R.P.J.
        • Bates M.
        • Stallinga S.
        • et al.
        Quantitative localization microscopy: effects of photophysics and labeling stoichiometry.
        PLoS One. 2015; 10: e0127989
        • Fricke F.
        • Beaudouin J.
        • Heilemann M.
        • et al.
        One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy.
        Sci. Rep. 2015; 5: 14072
        • Wang Y.
        • Penkul P.
        • Milstein J.N.
        Quantitative localization microscopy reveals a novel organization of a high-copy number plasmid.
        Biophys. J. 2016; 111: 467-479
        • Nino D.
        • Rafiei N.
        • Milstein J.N.
        • et al.
        Molecular counting with localization microscopy: a bayesian estimate based on fluorophore statistics.
        Biophys. J. 2017; 112: 1777-1785
        • Nino D.
        • Djayakarsana D.
        • Milstein J.N.
        Nanoscopic stoichiometry and single-molecule counting.
        Small Methods. 2019; 3: 1900082
        • Boonkird A.
        • Nino D.F.
        • Milstein J.N.
        An expectation-maximization approach to quantifying protein stoichiometry with single-molecule imaging.
        Bioinformatics Adv. 2021; 1https://doi.org/10.1093/bioadv/vbab032
        • Nino D.F.
        • Milstein J.N.
        Estimating the dynamic range of quantitative single-molecule localization microscopy.
        Biophys. J. 2021; 120: 3901-3910
        • van de Linde S.
        • Wolter S.
        • Sauer M.
        • et al.
        The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging.
        J. Biotechnol. 2010; 149: 260-266
        • Zhang H.
        • Guo P.
        Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation.
        Methods. 2014; 67: 169-176
        • Dey S.
        • Maiti S.
        Single-molecule photobleaching: instrumentation and applications.
        J. Biosci. 2018; 43: 447-454
        • Lelek M.
        • Gyparaki M.T.
        • Zimmer C.
        • et al.
        Single-molecule localization microscopy.
        Nat. Rev. Methods Primers. 2021; 1: 39-47
        • Vangindertael J.
        • Camacho R.
        • Janssen K.P.F.
        • et al.
        An introduction to optical super-resolution microscopy for the adventurous biologist.
        Methods Appl. Fluoresc. 2018; 6: 022003
        • Vogelsang J.
        • Kasper R.
        • Tinnefeld P.
        • et al.
        A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes.
        Angew. Chem. Int. Ed. Engl. 2008; 47: 5465-5469
        • Deschenes L.A.
        • Vanden Bout D.A.
        Single molecule photobleaching: increasing photon yield and survival time through suppression of two-step photolysis.
        Chem. Phys. Lett. 2002; 365: 387-395
        • Aitken C.E.
        • Marshall R.A.
        • Puglisi J.D.
        An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments.
        Biophys. J. 2008; 94: 1826-1835
        • Chen H.
        • Ahsan S.S.
        • Webb W.W.
        • et al.
        Mechanisms of quenching of Alexa fluorophores by natural amino acids.
        J. Am. Chem. Soc. 2010; 132: 7244-7245
        • Zhao Q.
        • Young I.T.
        • de Jong J.G.S.
        Photon budget analysis for fluorescence lifetime imaging microscopy.
        J. Biomed. Opt. 2011; 16: 086007
        • Wang S.
        • Moffitt J.R.
        • Zhuang X.
        • et al.
        Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging.
        Proc. Natl. Acad. Sci. USA. 2014; 111: 8452-8457
        • Krasowska J.
        • Olasek M.
        • Wielgus-Kutrowska B.
        • et al.
        The comparison of aggregation and folding of enhanced green fluorescent protein (EGFP) by spectroscopic studies.
        Spectroscopy. 2010; 24: 343-348
        • Heppert J.K.
        • Dickinson D.J.
        • Goldstein B.
        • et al.
        Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system.
        Mol. Biol. Cell. 2016; 27: 3385-3394
        • Subach F.V.
        • Patterson G.H.
        • Verkhusha V.V.
        • et al.
        Photoactivatable mCherry for high-resolution two-color fluorescence microscopy.
        Nat. Methods. 2009; 6: 153-159
        • Dave R.
        • Terry D.S.
        • Blanchard S.C.
        • et al.
        Mitigating unwanted photophysical processes for improved single-molecule fluorescence imaging.
        Biophys. J. 2009; 96: 2371-2381
        • Bharill S.
        • Chen C.
        • Goldman Y.E.
        • et al.
        Enhancement of single molecule fluorescence signals by colloidal silver nanoparticles in studies of protein translation.
        ACS Nano. 2011; 5: 399-407
        • Glembockyte V.
        • Lin J.
        • Cosa G.
        Improving the photostability of red- and green-emissive single-molecule fluorophores via Ni2+ mediated excited triplet-state quenching.
        J. Phys. Chem. B. 2016; 120: 11923-11929
        • Michie M.S.
        • Götz R.
        • Schnermann M.J.
        • et al.
        Cyanine conformational restraint in the far-red range.
        J. Am. Chem. Soc. 2017; 139: 12406-12409
        • Glembockyte V.
        • Wieneke R.
        • Cosa G.
        • et al.
        Tris -N-nitrilotriacetic acid fluorophore as a self-healing dye for single-molecule fluorescence imaging.
        J. Am. Chem. Soc. 2018; 140: 11006-11012
        • Chalfie M.
        • Tu Y.
        • Prasher D.C.
        • et al.
        Green fluorescent protein as a marker for gene expression.
        Science. 1994; 263: 802-805
        • Dickson R.M.
        • Cubitt A.B.
        • Moerner W.E.
        • et al.
        On/off blinking and switching behaviour of single molecules of green fluorescent protein.
        Nature. 1997; 388: 355-358
        • Shaner N.C.
        • Patterson G.H.
        • Davidson M.W.
        Advances in fluorescent protein technology.
        J. Cell Sci. 2007; 120: 4247-4260
        • Cranfill P.J.
        • Sell B.R.
        • Piston D.W.
        • et al.
        Quantitative assessment of fluorescent proteins.
        Nat. Methods. 2016; 13: 557-562
        • Ai H.W.
        • Baird M.A.
        • Campbell R.E.
        • et al.
        Engineering and characterizing monomeric fluorescent proteins for live-cell imaging applications.
        Nat. Protoc. 2014; 9: 910-928
        • Thevathasan J.V.
        • Kahnwald M.
        • Ries J.
        • et al.
        Nuclear pores as versatile reference standards for quantitative superresolution microscopy.
        Nat. Methods. 2019; 16: 1045-1053
        • Dunsing V.
        • Luckner M.
        • Chiantia S.
        • et al.
        Optimal fluorescent protein tags for quantifying protein oligomerization in living cells.
        Sci. Rep. 2018; 8: 10634
        • Juette M.F.
        • Terry D.S.
        • Blanchard S.C.
        • et al.
        The bright future of single-molecule fluorescence imaging.
        Curr. Opin. Chem. Biol. 2014; 20: 103-111
        • Zosel F.
        • Holla A.
        • Schuler B.
        Labeling of proteins for single-molecule fluorescence spectroscopy.
        in: Muñoz V. Protein Folding: Methods and Protocols. Springer US, 2022: 207-233
        • Becker C.F.W.
        • Seidel R.
        • Engelhard M.
        • et al.
        C-terminal fluorescence labeling of proteins for interaction studies on the single-molecule level.
        Chembiochem. 2006; 7: 891-895
        • Fernandes D.D.
        • Bamrah J.
        • Gradinaru C.C.
        • et al.
        Characterization of fluorescein arsenical hairpin (FlAsH) as a probe for single-molecule fluorescence spectroscopy.
        Sci. Rep. 2017; 7: 13063
        • Nikić I.
        • Kang J.H.
        • Lemke E.A.
        • et al.
        Labeling proteins on live mammalian cells using click chemistry.
        Nat. Protoc. 2015; 10: 780-791
        • Los G.V.
        • Encell L.P.
        • Wood K.V.
        • et al.
        HaloTag: a novel protein labeling technology for cell imaging and protein analysis.
        ACS Chem. Biol. 2008; 3: 373-382
        • Sun X.
        • Zhang A.
        • Corrêa Jr., I.R.
        • et al.
        Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging.
        Chembiochem. 2011; 12: 2217-2226
        • Gautier A.
        • Juillerat A.
        • Johnsson K.
        • et al.
        An engineered protein tag for multiprotein labeling in living cells.
        Chem. Biol. 2008; 15: 128-136
        • Blanchard S.C.
        • Gonzalez R.L.
        • Puglisi J.D.
        • et al.
        tRNA selection and kinetic proofreading in translation.
        Nat. Struct. Mol. Biol. 2004; 11: 1008-1014
        • Zhang M.
        • Chang H.
        • Xu T.
        • et al.
        Rational design of true monomeric and bright photoactivatable fluorescent proteins.
        Nat. Methods. 2012; 9: 727-729
        • Jradi F.M.
        • Lavis L.D.
        Chemistry of photosensitive fluorophores for single-molecule localization microscopy.
        ACS Chem. Biol. 2019; 14: 1077-1090
        • Li H.
        • Vaughan J.C.
        Switchable fluorophores for single-molecule localization microscopy.
        Chem. Rev. 2018; 118: 9412-9454
        • McEvoy A.L.
        • Hoi H.
        • Campbell R.E.
        • et al.
        mMaple: a photoconvertible fluorescent protein for use in multiple imaging modalities.
        PLoS One. 2012; 7: e51314
        • Subach F.V.
        • Patterson G.H.
        • Verkhusha V.V.
        • et al.
        Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.
        J. Am. Chem. Soc. 2010; 132: 6481-6491
        • Dempsey G.T.
        • Vaughan J.C.
        • Zhuang X.
        • et al.
        Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.
        Nat. Methods. 2011; 8: 1027-1036
        • Grimm J.B.
        • English B.P.
        • Lavis L.D.
        • et al.
        A general method to improve fluorophores for live-cell and single-molecule microscopy.
        Nat. Methods. 2015; 12: 244-250
        • Grimm J.B.
        • Xie L.
        • Lavis L.D.
        • et al.
        Deuteration improves small-molecule fluorophores.
        bioRxiv. 2020; (Preprint at)https://doi.org/10.1101/2020.08.17.250027
        • Nahidiazar L.
        • Agronskaia A.V.
        • Jalink K.
        • et al.
        Optimizing imaging conditions for demanding multi-color super resolution localization microscopy.
        PLoS One. 2016; 11: e0158884
        • Olivier N.
        • Keller D.
        • Manley S.
        • et al.
        Resolution doubling in 3D-STORM imaging through improved buffers.
        PLoS One. 2013; 8: e69004
        • van der Velde J.H.M.
        • Smit J.H.
        • Cordes T.
        • et al.
        Self-healing dyes for super-resolution fluorescence microscopy.
        J. Phys. D Appl. Phys. 2018; 52: 034001
        • Tinnefeld P.
        • Cordes T.
        “Self-healing” dyes: intramolecular stabilization of organic fluorophores.
        Nat. Methods. 2012; 9 (author reply 427-428): 426-427
        • Isselstein M.
        • Zhang L.
        • Cordes T.
        • et al.
        Self-healing dyes—keeping the promise?.
        J. Phys. Chem. Lett. 2020; 11: 4462-4480
        • Uno S.N.
        • Kamiya M.
        • Urano Y.
        • et al.
        A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging.
        Nat. Chem. 2014; 6: 681-689
        • Erdmann R.S.
        • Baguley S.W.
        • Toomre D.
        • et al.
        Labeling strategies matter for super-resolution microscopy: a comparison between HaloTags and SNAP-tags.
        Cell Chem. Biol. 2019; 26: 584-592.e6
        • Axelrod D.
        Chapter 7 total internal reflection fluorescence microscopy.
        in: Methods in Cell Biology. Academic Press, 2008: 169-221
        • Wu P.-H.
        • Nelson N.
        • Tseng Y.
        A general method for improving spatial resolution by optimization of electron multiplication in CCD imaging.
        Opt. Express. 2010; 18: 5199-5212
        • Robbins M.S.
        • Hadwen B.J.
        The noise performance of electron multiplying charge-coupled devices.
        IEEE Trans. Electron. Devices. 2003; 50: 1227-1232
        • Bigas M.
        • Cabruja E.
        • Salvi J.
        • et al.
        Review of CMOS image sensors.
        Microelectron. J. 2006; 37: 433-451
        • Wang Y.
        • Zhao L.
        • Huang Z.-L.
        • et al.
        Quantitative performance evaluation of a back-illuminated sCMOS camera with 95% QE for super-resolution localization microscopy.
        Cytometry A. 2017; 91: 1175-1183
        • Almada P.
        • Culley S.
        • Henriques R.
        PALM and STORM: into large fields and high-throughput microscopy with sCMOS detectors.
        Methods. 2015; 88: 109-121
        • Khaw I.
        • Croop B.
        • Han K.Y.
        • et al.
        Flat-field illumination for quantitative fluorescence imaging.
        Opt. Express. 2018; 26: 15276-15288
        • Lam J.Y.L.
        • Wu Y.
        • Danial J.S.H.
        • et al.
        An economic, square-shaped flat-field illumination module for TIRF-based super-resolution microscopy.
        Biophys. Rep. 2021; https://doi.org/10.1016/j.bpr.2022.100044
        • Ibrahim K.A.
        • Mahecic D.
        • Manley S.
        Characterization of flat-fielding systems for quantitative microscopy.
        Opt. Express. 2020; 28: 22036-22048
        • Herdly L.
        • Janin P.
        • van de Linde S.
        • et al.
        Tunable wide-field illumination and single-molecule photoswitching with a single MEMS mirror.
        ACS Photonics. 2021; 8: 2728-2736
        • Mau A.
        • Friedl K.
        • Lévêque-Fort S.
        • et al.
        Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields.
        Nat. Commun. 2021; 12: 3077
        • Diekmann R.
        • Till K.
        • Huser T.
        • et al.
        Characterization of an industry-grade CMOS camera well suited for single molecule localization microscopy – high performance super-resolution at low cost.
        Sci. Rep. 2017; 7: 14425
        • Golfetto O.
        • Wakefield D.L.
        • Jovanović-Talisman T.
        • et al.
        A platform to enhance quantitative single molecule localization microscopy.
        J. Am. Chem. Soc. 2018; 140: 12785-12797
        • Zanacchi F.C.
        • Manzo C.
        • Lakadamyali M.
        • et al.
        A DNA origami platform for quantifying protein copy number in super-resolution.
        Nat. Methods. 2017; 14: 789-792
        • Scheckenbach M.
        • Bauer J.
        • Tinnefeld P.
        • et al.
        DNA origami nanorulers and emerging reference structures.
        Apl. Mater. 2020; 8: 110902
        • Lin R.
        • Clowsley A.H.
        • Soeller C.
        • et al.
        3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples.
        Methods. 2020; 174: 56-71
        • Schmied J.J.
        • Raab M.
        • Tinnefeld P.
        • et al.
        DNA origami–based standards for quantitative fluorescence microscopy.
        Nat. Protoc. 2014; 9: 1367-1391
        • Calebiro D.
        • Rieken F.
        • Lohse M.J.
        • et al.
        Single-molecule analysis of fluorescently labeled G-protein–coupled receptors reveals complexes with distinct dynamics and organization.
        Proc. Natl. Acad. Sci. USA. 2013; 110: 743-748
        • Durisic N.
        • Laparra-Cuervo L.
        • Lakadamyali M.
        • et al.
        Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate.
        Nat. Methods. 2014; 11: 156-162
        • Finan K.
        • Raulf A.
        • Heilemann M.
        A set of homo-oligomeric standards allows accurate protein counting.
        Angew. Chem. Int. Ed. Engl. 2015; 54: 12049-12052
        • Chidiac P.
        • Green M.A.
        • Wells J.W.
        • et al.
        Cardiac muscarinic receptors. Cooperativity as the basis for multiple states of affinity.
        Biochemistry. 1997; 36: 7361-7379
        • Li Y.
        • Shivnaraine R.V.
        • Gradinaru C.C.
        • et al.
        Ligand-induced coupling between oligomers of the M2 receptor and the Gi1 protein in live cells.
        Biophys. J. 2018; 115: 881-895
        • Işbilir A.
        • Möller J.
        • Lohse M.J.
        • et al.
        Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists.
        Proc. Natl. Acad. Sci. USA. 2020; 117: 29144-29154
        • Levitz J.
        • Habrian C.
        • Isacoff E.Y.
        • et al.
        Mechanism of assembly and cooperativity of homomeric and heteromeric metabotropic glutamate receptors.
        Neuron. 2016; 92: 143-159
        • Jonas K.C.
        • Fanelli F.
        • Hanyaloglu A.C.
        • et al.
        Single molecule analysis of functionally asymmetric G protein-coupled receptor (GPCR) oligomers reveals diverse spatial and structural assemblies.
        J. Biol. Chem. 2015; 290: 3875-3892
        • Siddig S.
        • Aufmkolk S.
        • Calebiro D.
        • et al.
        Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones.
        Sci. Adv. 2020; 6: eaay7193
        • Möller J.
        • Isbilir A.
        • Lohse M.J.
        • et al.
        Single-molecule analysis reveals agonist-specific dimer formation of μ-opioid receptors.
        Nat. Chem. Biol. 2020; 16: 946-954
        • Joseph M.D.
        • Tomas Bort E.
        • Simoncelli S.
        • et al.
        Quantitative super-resolution imaging for the analysis of GPCR oligomerization.
        Biomolecules. 2021; 11: 1503
        • He H.
        • Liu L.
        • Huang F.
        • et al.
        Carbon dot blinking enables accurate molecular counting at nanoscale resolution.
        Anal. Chem. 2021; 93: 3968-3975
        • Wisler J.W.
        • Xiao K.
        • Lefkowitz R.J.
        • et al.
        Recent developments in biased agonism.
        Curr. Opin. Cell Biol. 2014; 27: 18-24