Analyzing Protein Clusters on the Plasma Membrane: Application of Spatial Statistical Analysis Methods on Super-Resolution Microscopy Images

  • Laura Paparelli
  • Nikky Corthout
  • Benjamin Pavie
  • Wim Annaert
  • Sebastian MunckEmail author
Part of the Advances in Anatomy, Embryology and Cell Biology book series (ADVSANAT, volume 219)


The spatial distribution of proteins within the cell affects their capability to interact with other molecules and directly influences cellular processes and signaling. At the plasma membrane, multiple factors drive protein compartmentalization into specialized functional domains, leading to the formation of clusters in which intermolecule interactions are facilitated. Therefore, quantifying protein distributions is a necessity for understanding their regulation and function. The recent advent of super-resolution microscopy has opened up the possibility of imaging protein distributions at the nanometer scale. In parallel, new spatial analysis methods have been developed to quantify distribution patterns in super-resolution images. In this chapter, we provide an overview of super-resolution microscopy and summarize the factors influencing protein arrangements on the plasma membrane. Finally, we highlight methods for analyzing clusterization of plasma membrane proteins, including examples of their applications.


Pair Correlation Function Plasma Membrane Protein Immunological Synapse Complete Spatial Randomness Spatial Statistical Analysis 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors would like to thank Dr. Susana Rocha and Dr. Vinoy Vijayan for their fruitful discussion and Dr. Donna Stolz for the silica particles to isolate plasma membranes. This work is financially supported by VIB, VIB Bio Imaging Core facility, the Hercules Foundation for heavy infrastructure (Hercules AKUL058/HER/08/021, AKUL/09/037, and AKUL13/39 (ISPAMM)), KU Leuven (IDO/12/020), the federal government (IAP P7/16), and SAO-FRA (S#14017). SM was supported by a grant from KU Leuven (CREA/12/22).


  1. Abbe E (1873) Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Arch Für Mikrosk Anat 9:413–418. doi: 10.1007/BF02956173 CrossRefGoogle Scholar
  2. Adam V, Moeyaert B, David CC et al (2011) Rational design of photoconvertible and biphotochromic fluorescent proteins for advanced microscopy applications. Chem Biol 18:1241–1251. doi: 10.1016/j.chembiol.2011.08.007 PubMedCrossRefGoogle Scholar
  3. Annibale P, Vanni S, Scarselli M et al (2011) Identification of clustering artifacts in photoactivated localization microscopy. Nat Methods 8:527–528. doi: 10.1038/nmeth.1627 PubMedCrossRefGoogle Scholar
  4. Aoyagi K, Sugaya T, Umeda M et al (2005) The activation of exocytotic sites by the formation of phosphatidylinositol 4,5-bisphosphate microdomains at syntaxin clusters. J Biol Chem 280:17346–17352. doi: 10.1074/jbc.M413307200 PubMedCrossRefGoogle Scholar
  5. Baddeley A (2007) Spatial point processes and their applications. In: Stochastic geometry. Springer, Berlin Heidelberg, pp 1–75Google Scholar
  6. Baddeley A, Turner R (2005) spatstat: An R package for analyzing spatial point patterns. J Stat Softw 12(6). doi: 10.18637/jss.v012.i06
  7. Baddeley D, Cannell MB, Soeller C (2011) Three-dimensional sub-100 nm super-resolution imaging of biological samples using a phase ramp in the objective pupil. Nano Res 4:589–598. doi: 10.1007/s12274-011-0115-z CrossRefGoogle Scholar
  8. Balagopalan L, Barr VA, Kortum RL et al (2013) Cutting edge: cell surface linker for activation of T cells is recruited to microclusters and is active in signaling. J Immunol Baltim Md 190:3849–3853. doi: 10.4049/jimmunol.1202760 Google Scholar
  9. Bar-On D, Wolter S, van de Linde S et al (2012) Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters. J Biol Chem 287:27158–27167. doi: 10.1074/jbc.M112.353250 PubMedPubMedCentralCrossRefGoogle Scholar
  10. Barreiro O, Zamai M, Yáñez-Mó M et al (2008) Endothelial adhesion receptors are recruited to adherent leukocytes by inclusion in preformed tetraspanin nanoplatforms. J Cell Biol 183:527–542. doi: 10.1083/jcb.200805076 PubMedPubMedCentralCrossRefGoogle Scholar
  11. Belardi B, O’Donoghue GP, Smith AW et al (2012) Investigating cell surface galectin-mediated cross-linking on glycoengineered cells. J Am Chem Soc 134:9549–9552. doi: 10.1021/ja301694s PubMedPubMedCentralCrossRefGoogle Scholar
  12. Betzig E, Chichester RJ (1993) Single molecules observed by near-field scanning optical microscopy. Science 262:1422–1425. doi: 10.1126/science.262.5138.1422 PubMedCrossRefGoogle Scholar
  13. Betzig E, Trautman JK (1992) Near-field optics: microscopy, spectroscopy, and surface modification beyond the diffraction limit. Science 257:189–195. doi: 10.1126/science.257.5067.189 PubMedCrossRefGoogle Scholar
  14. Betzig E, Patterson GH, Sougrat R et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645. doi: 10.1126/science.1127344 PubMedCrossRefGoogle Scholar
  15. Blom H, RöNnlund D, Scott L et al (2012) Nearest neighbor analysis of dopamine D1 receptors and Na+-K+-ATPases in dendritic spines dissected by STED microscopy. Microsc Res Tech 75:220–228. doi: 10.1002/jemt.21046 PubMedCrossRefGoogle Scholar
  16. Bolte S, Cordelières FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224:213–232. doi: 10.1111/j.1365-2818.2006.01706.x PubMedCrossRefGoogle Scholar
  17. Boscher C, Dennis JW, Nabi IR (2011) Glycosylation, galectins and cellular signaling. Curr Opin Cell Biol 23:383–392. doi: 10.1016/ PubMedCrossRefGoogle Scholar
  18. Boucheix C, Rubinstein E (2001) Tetraspanins. Cell Mol Life Sci CMLS 58:1189–1205PubMedCrossRefGoogle Scholar
  19. Brewer CF, Miceli MC, Baum LG (2002) Clusters, bundles, arrays and lattices: novel mechanisms for lectin–saccharide-mediated cellular interactions. Curr Opin Struct Biol 12:616–623. doi: 10.1016/S0959-440X(02)00364-0 PubMedCrossRefGoogle Scholar
  20. Brown DA, Rose JK (1992) Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68:533–544PubMedCrossRefGoogle Scholar
  21. Burgert A, Letschert S, Doose S, Sauer M (2015) Artifacts in single-molecule localization microscopy. Histochem Cell Biol 144:123–131. doi: 10.1007/s00418-015-1340-4 PubMedCrossRefGoogle Scholar
  22. Burt JE, Barber GM, Rigby DL (2009) Elementary statistics for geographers, 3rd edn. Guilford Press, New YorkGoogle Scholar
  23. Cardona A, Saalfeld S, Preibisch S et al (2010) An integrated micro- and macroarchitectural analysis of the drosophila brain by computer-assisted serial section electron microscopy. PLoS Biol 8, e1000502. doi: 10.1371/journal.pbio.1000502 PubMedPubMedCentralCrossRefGoogle Scholar
  24. Chaney LK, Jacobson BS (1983) Coating cells with colloidal silica for high yield isolation of plasma membrane sheets and identification of transmembrane proteins. J Biol Chem 258:10062–10072PubMedGoogle Scholar
  25. Chaudhary N, Gomez GA, Howes MT et al (2014) Endocytic crosstalk: cavins, caveolins, and caveolae regulate clathrin-independent endocytosis. PLoS Biol 12, e1001832. doi: 10.1371/journal.pbio.1001832 PubMedPubMedCentralCrossRefGoogle Scholar
  26. Constals A, Penn AC, Compans B et al (2015) Glutamate-induced AMPA receptor desensitization increases their mobility and modulates short-term plasticity through unbinding from stargazin. Neuron 85:787–803. doi: 10.1016/j.neuron.2015.01.012 PubMedCrossRefGoogle Scholar
  27. Cox S, Rosten E, Monypenny J et al (2012) Bayesian localization microscopy reveals nanoscale podosome dynamics. Nat Methods 9:195–200. doi: 10.1038/nmeth.1812 CrossRefGoogle Scholar
  28. Cressie ACN (1993) Statistics for spatial data. Wiley and Sons, New York, Revised EditionGoogle Scholar
  29. de Bakker BI, de Lange F, Cambi A et al (2007) Nanoscale organization of the pathogen receptor DC-SIGN mapped by single-molecule high-resolution fluorescence microscopy. Chem Phys Chem 8:1473–1480. doi: 10.1002/cphc.200700169 PubMedGoogle Scholar
  30. de Lange F, Cambi A, Huijbens R et al (2001) Cell biology beyond the diffraction limit: near-field scanning optical microscopy. J Cell Sci 114:4153–4160PubMedGoogle Scholar
  31. Dertinger T, Colyer R, Iyer G et al (2009) Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc Natl Acad Sci U S A 106:22287–22292. doi: 10.1073/pnas.0907866106 PubMedPubMedCentralCrossRefGoogle Scholar
  32. Deschout H, Zanacchi FC, Mlodzianoski M et al (2014a) Precisely and accurately localizing single emitters in fluorescence microscopy. Nat Methods 11:253–266. doi: 10.1038/nmeth.2843 PubMedCrossRefGoogle Scholar
  33. Deschout H, Shivanandan A, Annibale P et al (2014b) Progress in quantitative single-molecule localization microscopy. Histochem Cell Biol 142:5–17. doi: 10.1007/s00418-014-1217-y PubMedPubMedCentralCrossRefGoogle Scholar
  34. Diaz-Rohrer B, Levental KR, Levental I (2014) Rafting through traffic: membrane domains in cellular logistics. Biochim Biophys Acta BBA – Biomembr 1838:3003–3013. doi: 10.1016/j.bbamem.2014.07.029 CrossRefGoogle Scholar
  35. Dixon PM (2002) Ripley’s K function. In: El-Shaarawi AH, Piegorsch WW (eds) Encyclopedia of environmetrics. John Wiley & Sons, Ltd, Chichester, pp 1796–1803Google Scholar
  36. Donnert G, Keller J, Wurm CA et al (2007) Two-color far-field fluorescence nanoscopy. Biophys J 92:L67–L69. doi: 10.1529/biophysj.107.104497 PubMedPubMedCentralCrossRefGoogle Scholar
  37. Ehrig J, Petrov EP, Schwille P (2011) Near-critical fluctuations and cytoskeleton-assisted phase separation lead to subdiffusion in cell membranes. Biophys J 100:80–89. doi: 10.1016/j.bpj.2010.11.002 PubMedPubMedCentralCrossRefGoogle Scholar
  38. Endesfelder U, Heilemann M (2014) Art and artifacts in single-molecule localization microscopy: beyond attractive images. Nat Methods 11:235–238. doi: 10.1038/nmeth.2852 PubMedCrossRefGoogle Scholar
  39. Espenel C, Margeat E, Dosset P et al (2008) Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web. J Cell Biol 182:765–776. doi: 10.1083/jcb.200803010 PubMedPubMedCentralCrossRefGoogle Scholar
  40. Ester M, Kriegel H, Sander J, Xu X (1996) A density-based algorithm for discovering clusters in large spatial databases with noise. AAAI Press, Palo Alta, pp 226–231. doi:  10.1023/A:1009745219419
  41. Feigenson GW (2007) Phase boundaries and biological membranes. Annu Rev Biophys Biomol Struct 36:63–77. doi: 10.1146/annurev.biophys.36.040306.132721 PubMedPubMedCentralCrossRefGoogle Scholar
  42. Flors C, Hotta J, Uji-i H et al (2007) A stroboscopic approach for fast photoactivation−localization microscopy with dronpa mutants. J Am Chem Soc 129:13970–13977. doi: 10.1021/ja074704l PubMedCrossRefGoogle Scholar
  43. Fölling J, Bossi M, Bock H et al (2008) Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods 5:943–945. doi: 10.1038/nmeth.1257 PubMedCrossRefGoogle Scholar
  44. Fornasiero EF, Rizzoli SO (2014) Super-resolution microscopy techniques in the neurosciences. Humana Press, TotowaCrossRefGoogle Scholar
  45. Friedl P, den Boer AT, Gunzer M (2005) Tuning immune responses: diversity and adaptation of the immunological synapse. Nat Rev Immunol 5:532–545. doi: 10.1038/nri1647 PubMedCrossRefGoogle Scholar
  46. Fujiwara T, Ritchie K, Murakoshi H et al (2002) Phospholipids undergo hop diffusion in compartmentalized cell membrane. J Cell Biol 157:1071–1082. doi: 10.1083/jcb.200202050 PubMedPubMedCentralCrossRefGoogle Scholar
  47. Gambin Y, Ariotti N, McMahon K-A et al (2014) Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae. eLife 3, e01434. doi: 10.7554/eLife.01434 PubMedCentralCrossRefGoogle Scholar
  48. Geisler C, Schönle A, von Middendorff C et al (2007) Resolution of λ /10 in fluorescence microscopy using fast single molecule photo-switching. Appl Phys A 88:223–226. doi: 10.1007/s00339-007-4144-0 CrossRefGoogle Scholar
  49. Getis A, Ord JK (1992) The analysis of spatial association by use of distance statistics. Geogr Anal 24:189–206. doi: 10.1111/j.1538-4632.1992.tb00261.x CrossRefGoogle Scholar
  50. Godin AG, Costantino S, Lorenzo L-E et al (2011) Revealing protein oligomerization and densities in situ using spatial intensity distribution analysis. Proc Natl Acad Sci 108:7010–7015. doi: 10.1073/pnas.1018658108 PubMedPubMedCentralCrossRefGoogle Scholar
  51. Gong W, Si K, Chen N, Sheppard CJR (2010) Focal modulation microscopy with annular apertures: a numerical study. J Biophotonics 3:476–484. doi: 10.1002/jbio.200900110 PubMedCrossRefGoogle Scholar
  52. Gorter E, Grendel F (1925) On bimolecular layers of lipoids on the chromocytes of the blood. J Exp Med 41:439–443PubMedPubMedCentralCrossRefGoogle Scholar
  53. Greig-Smith P (1952) The Use of random and contiguous quadrats in the study of the structure of plant communities. Ann Bot 16:293–316Google Scholar
  54. Gudheti MV, Curthoys NM, Gould TJ et al (2013) Actin mediates the nanoscale membrane organization of the clustered membrane protein influenza hemagglutinin. Biophys J 104:2182–2192. doi: 10.1016/j.bpj.2013.03.054 PubMedPubMedCentralCrossRefGoogle Scholar
  55. Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198:82–87PubMedCrossRefGoogle Scholar
  56. Gustafsson MGL (2005) Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci U S A 102:13081–13086. doi: 10.1073/pnas.0406877102 PubMedPubMedCentralCrossRefGoogle Scholar
  57. Gustafsson MGL, Shao L, Carlton PM et al (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94:4957–4970. doi: 10.1529/biophysj.107.120345 PubMedPubMedCentralCrossRefGoogle Scholar
  58. Hamel V, Guichard P, Fournier M et al (2014) Correlative multicolor 3D SIM and STORM microscopy. Biomed Opt Exp 5:3326–3336. doi: 10.1364/BOE.5.003326 CrossRefGoogle Scholar
  59. Haque U, Overgaard HJ, Clements ACA et al (2014) Malaria burden and control in Bangladesh and prospects for elimination: an epidemiological and economic assessment. Lancet Glob Health 2:e98–e105. doi: 10.1016/S2214-109X(13)70176-1 PubMedCrossRefGoogle Scholar
  60. Heilemann M, van de Linde S, Schüttpelz M et al (2008) Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed 47:6172–6176. doi: 10.1002/anie.200802376 CrossRefGoogle Scholar
  61. Heilemann M, van de Linde S, Mukherjee A, Sauer M (2009) Super-resolution imaging with small organic fluorophores. Angew Chem Int Ed Engl 48:6903–6908. doi: 10.1002/anie.200902073 PubMedCrossRefGoogle Scholar
  62. Hein B, Willig KI, Hell SW (2008) Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell. Proc Natl Acad Sci 105:14271–14276. doi: 10.1073/pnas.0807705105 PubMedPubMedCentralCrossRefGoogle Scholar
  63. Heintzmann R (2003) Saturated patterned excitation microscopy with two-dimensional excitation patterns. Micron Oxf Engl 34:283–291Google Scholar
  64. Heintzmann R, Cremer CG (1999) Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. Proc SPIE 3568:185–196. doi:  10.1117/12.336833
  65. Heintzmann R, Jovin TM, Cremer C (2002) Saturated patterned excitation microscopy--a concept for optical resolution improvement. J Opt Soc Am A Opt Image Sci Vis 19:1599–1609PubMedCrossRefGoogle Scholar
  66. Hell SW, Kroug M (1995) Ground-state-depletion fluorescence microscopy: a concept for breaking the diffraction resolution limit. Appl Phys B 60:495–497. doi: 10.1007/BF01081333 CrossRefGoogle Scholar
  67. Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19:780–782PubMedCrossRefGoogle Scholar
  68. Helmuth JA, Paul G, Sbalzarini IF (2010) Beyond co-localization: inferring spatial interactions between sub-cellular structures from microscopy images. BMC Bioinformatics 11:1–12. doi: 10.1186/1471-2105-11-372 CrossRefGoogle Scholar
  69. Hemler ME (2003) Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. Annu Rev Cell Dev Biol 19:397–422. doi: 10.1146/annurev.cellbio.19.111301.153609 PubMedCrossRefGoogle Scholar
  70. Hemler ME (2005) Tetraspanin functions and associated microdomains. Nat Rev Mol Cell Biol 6:801–811. doi: 10.1038/nrm1736 PubMedCrossRefGoogle Scholar
  71. Henriques R, Lelek M, Fornasiero EF et al (2010) QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ. Nat Methods 7:339–340. doi: 10.1038/nmeth0510-339 PubMedCrossRefGoogle Scholar
  72. Hess ST, Girirajan TPK, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91:4258–4272. doi: 10.1529/biophysj.106.091116 PubMedPubMedCentralCrossRefGoogle Scholar
  73. Honigmann A, van den Bogaart G, Iraheta E et al (2013) Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment. Nat Struct Mol Biol 20:679–686. doi: 10.1038/nsmb.2570 PubMedPubMedCentralCrossRefGoogle Scholar
  74. Horejsi V, Hrdinka M (2014) Membrane microdomains in immunoreceptor signaling. FEBS Lett 588:2392–2397. doi: 10.1016/j.febslet.2014.05.047 PubMedCrossRefGoogle Scholar
  75. Hoze N, Nair D, Hosy E et al (2012) Heterogeneity of AMPA receptor trafficking and molecular interactions revealed by superresolution analysis of live cell imaging. Proc Natl Acad Sci 109:17052–17057. doi: 10.1073/pnas.1204589109 PubMedPubMedCentralCrossRefGoogle Scholar
  76. Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319:810–813. doi: 10.1126/science.1153529 PubMedPubMedCentralCrossRefGoogle Scholar
  77. Ipsen JH, Karlström G, Mouritsen OG et al (1987) Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim Biophys Acta 905:162–172PubMedCrossRefGoogle Scholar
  78. Itano MS, Steinhauer C, Schmied JJ et al (2012) Super-resolution imaging of C-type lectin and influenza hemagglutinin nanodomains on plasma membranes using blink microscopy. Biophys J 102:1534–1542. doi: 10.1016/j.bpj.2012.02.022 PubMedPubMedCentralCrossRefGoogle Scholar
  79. James DJ, Khodthong C, Kowalchyk JA, Martin TFJ (2008) Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion. J Cell Biol 182:355–366. doi: 10.1083/jcb.200801056 PubMedPubMedCentralCrossRefGoogle Scholar
  80. Juette MF, Gould TJ, Lessard MD et al (2008) Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples. Nat Methods 5:527–529. doi: 10.1038/nmeth.1211 PubMedCrossRefGoogle Scholar
  81. Kellner RR, Baier CJ, Willig KI et al (2007) Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy. Neuroscience 144:135–143. doi: 10.1016/j.neuroscience.2006.08.071 PubMedCrossRefGoogle Scholar
  82. Khuong TM, Habets RLP, Kuenen S et al (2013) Synaptic PI(3,4,5)P3 is required for Syntaxin1A clustering and neurotransmitter release. Neuron 77:1097–1108. doi: 10.1016/j.neuron.2013.01.025 PubMedCrossRefGoogle Scholar
  83. Kusumi A, Sako Y (1996) Cell surface organization by the membrane skeleton. Curr Opin Cell Biol 8:566–574. doi: 10.1016/S0955-0674(96)80036-6 PubMedCrossRefGoogle Scholar
  84. Kusumi A, Sako Y, Yamamoto M (1993) Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys J 65:2021–2040PubMedPubMedCentralCrossRefGoogle Scholar
  85. Kusumi A, Suzuki KGN, Kasai RS et al (2011) Hierarchical mesoscale domain organization of the plasma membrane. Trends Biochem Sci 36:604–615. doi: 10.1016/j.tibs.2011.08.001 PubMedCrossRefGoogle Scholar
  86. Kusumi A, Tsunoyama TA, Hirosawa KM et al (2014) Tracking single molecules at work in living cells. Nat Chem Biol 10:524–532. doi: 10.1038/nchembio.1558 PubMedCrossRefGoogle Scholar
  87. Lagache T, Lang G, Sauvonnet N, Olivo-Marin J-C (2013) Analysis of the spatial organization of molecules with robust statistics. PLoS One 8, e80914. doi: 10.1371/journal.pone.0080914 PubMedPubMedCentralCrossRefGoogle Scholar
  88. Lakshminarayan R, Wunder C, Becken U et al (2014) Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers. Nat Cell Biol 16:592–603. doi: 10.1038/ncb2970 CrossRefGoogle Scholar
  89. Lang T, Rizzoli SO (2010) Membrane protein clusters at nanoscale resolution: more than pretty pictures. Phys Chem Chem Phys 25:116–124. doi: 10.1152/physiol.00044.2009 Google Scholar
  90. Lehmann M, Rocha S, Mangeat B et al (2011) Quantitative multicolor super-resolution microscopy reveals tetherin HIV-1 interaction. PLoS Pathog 7, e1002456. doi: 10.1371/journal.ppat.1002456 PubMedPubMedCentralCrossRefGoogle Scholar
  91. Levental I, Lingwood D, Grzybek M et al (2010) Palmitoylation regulates raft affinity for the majority of integral raft proteins. Proc Natl Acad Sci U S A 107:22050–22054. doi: 10.1073/pnas.1016184107 PubMedPubMedCentralCrossRefGoogle Scholar
  92. Li R, Gundersen GG (2008) Beyond polymer polarity: how the cytoskeleton builds a polarized cell. Nat Rev Mol Cell Biol 9:860–873. doi: 10.1038/nrm2522 PubMedCrossRefGoogle Scholar
  93. Lidke K, Rieger B, Jovin T, Heintzmann R (2005) Superresolution by localization of quantum dots using blinking statistics. Opt Express 13:7052–7062PubMedCrossRefGoogle Scholar
  94. Lillemeier BF, Mörtelmaier MA, Forstner MB et al (2010) TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation. Nat Immunol 11:90–96. doi: 10.1038/ni.1832 PubMedPubMedCentralCrossRefGoogle Scholar
  95. London E (2002) Insights into lipid raft structure and formation from experiments in model membranes. Curr Opin Struct Biol 12:480–486. doi: 10.1016/S0959-440X(02)00351-2 PubMedCrossRefGoogle Scholar
  96. Ludwig A, Howard G, Mendoza-Topaz C et al (2013) Molecular composition and ultrastructure of the caveolar coat complex. PLoS Biol 11, e1001640. doi: 10.1371/journal.pbio.1001640 PubMedPubMedCentralCrossRefGoogle Scholar
  97. MacGillavry HD, Song Y, Raghavachari S, Blanpied TA (2013) Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78:615–622. doi: 10.1016/j.neuron.2013.03.009 PubMedPubMedCentralCrossRefGoogle Scholar
  98. Malissen B, Grégoire C, Malissen M, Roncagalli R (2014) Integrative biology of T cell activation. Nat Immunol 15:790–797. doi: 10.1038/ni.2959 PubMedCrossRefGoogle Scholar
  99. Mandula O, Šestak IŠ, Heintzmann R, Williams CKI (2014) Localisation microscopy with quantum dots using non-negative matrix factorisation. Opt Express 22:24594–24605PubMedCrossRefGoogle Scholar
  100. Marin R, Rojo JA, Fabelo N et al (2013) Lipid raft disarrangement as a result of neuropathological progresses: a novel strategy for early diagnosis? Neuroscience 245:26–39. doi: 10.1016/j.neuroscience.2013.04.025 PubMedCrossRefGoogle Scholar
  101. Meister M, Tikkanen R (2014) Endocytic trafficking of membrane-bound cargo: a flotillin point of view. Membranes 4:356–371. doi: 10.3390/membranes4030356 PubMedPubMedCentralCrossRefGoogle Scholar
  102. Milovanovic D, Jahn R (2015) Organization and dynamics of SNARE proteins in the presynaptic membrane. Front Physiol 6:89. doi: 10.3389/fphys.2015.00089 PubMedPubMedCentralCrossRefGoogle Scholar
  103. Mollinedo F, Gajate C (2015) Lipid rafts as major platforms for signaling regulation in cancer. Adv Biol Regul 57:130–146. doi: 10.1016/j.jbior.2014.10.003 PubMedCrossRefGoogle Scholar
  104. Munck S, Miskiewicz K, Sannerud R et al (2012) Sub-diffraction imaging on standard microscopes through photobleaching microscopy with non-linear processing. J Cell Sci 125:2257–2266. doi: 10.1242/jcs.098939 PubMedCrossRefGoogle Scholar
  105. Nair D, Hosy E, Petersen JD et al (2013) Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95. J Neurosci 33:13204–13224. doi: 10.1523/JNEUROSCI.2381-12.2013 PubMedCrossRefGoogle Scholar
  106. Nawaz S, Heindl A, Koelble K, Yuan Y (2015) Beyond immune density: critical role of spatial heterogeneity in estrogen receptor-negative breast cancer. Mod Pathol Off J U S Can Acad Pathol Inc. doi: 10.1038/modpathol.2015.37 Google Scholar
  107. Nicolson GL (2014) The fluid-mosaic model of membrane structure: still relevant to understanding the structure, function and dynamics of biological membranes after more than 40 years. Biochim Biophys Acta 1838:1451–1466. doi: 10.1016/j.bbamem.2013.10.019 PubMedCrossRefGoogle Scholar
  108. Nieuwenhuizen RPJ, Lidke KA, Bates M et al (2013) Measuring image resolution in optical nanoscopy. Nat Methods 10:557–562. doi: 10.1038/nmeth.2448 PubMedPubMedCentralCrossRefGoogle Scholar
  109. Notelaers K, Rocha S, Paesen R et al (2014) Membrane distribution of the glycine receptor α3 studied by optical super-resolution microscopy. Histochem Cell Biol 142:79–90. doi: 10.1007/s00418-014-1197-y PubMedCrossRefGoogle Scholar
  110. Okabe S (2007) Molecular anatomy of the postsynaptic density. Mol Cell Neurosci 34:503–518. doi: 10.1016/j.mcn.2007.01.006 PubMedCrossRefGoogle Scholar
  111. Otto GP, Nichols BJ (2011) The roles of flotillin microdomains--endocytosis and beyond. J Cell Sci 124:3933–3940. doi: 10.1242/jcs.092015 PubMedCrossRefGoogle Scholar
  112. Palade GE (1953) Fine structure of blood capillaries. J Appl Phys 24:1424Google Scholar
  113. Parton RG, del Pozo MA (2013) Caveolae as plasma membrane sensors, protectors and organizers. Nat Rev Mol Cell Biol 14:98–112. doi: 10.1038/nrm3512 PubMedCrossRefGoogle Scholar
  114. Parton RG, Simons K (2007) The multiple faces of caveolae. Nat Rev Mol Cell Biol 8:185–194. doi: 10.1038/nrm2122 PubMedCrossRefGoogle Scholar
  115. Pavani SRP, Thompson MA, Biteen JS et al (2009) Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc Natl Acad Sci U S A 106:2995–2999. doi: 10.1073/pnas.0900245106 PubMedPubMedCentralCrossRefGoogle Scholar
  116. Pelkmans L, Bürli T, Zerial M, Helenius A (2004) Caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane traffic. Cell 118:767–780. doi: 10.1016/j.cell.2004.09.003 PubMedCrossRefGoogle Scholar
  117. Pertsinidis A, Mukherjee K, Sharma M et al (2013) Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ. Proc Natl Acad Sci 110:E2812–E2820. doi: 10.1073/pnas.1310654110 PubMedPubMedCentralCrossRefGoogle Scholar
  118. Peters KR, Carley WW, Palade GE (1985) Endothelial plasmalemmal vesicles have a characteristic striped bipolar surface structure. J Cell Biol 101:2233–2238PubMedCrossRefGoogle Scholar
  119. Pike LJ (2006) Rafts defined: a report on the Keystone symposium on lipid rafts and cell function. J Lipid Res 47:1597–1598. doi: 10.1194/jlr.E600002-JLR200 PubMedCrossRefGoogle Scholar
  120. Ramadoss J, Pastore MB, Magness RR (2013) Endothelial caveolar subcellular domain regulation of endothelial nitric oxide synthase. Clin Exp Pharmacol Physiol 40:753–764. doi: 10.1111/1440-1681.12136 PubMedPubMedCentralCrossRefGoogle Scholar
  121. Rayleigh L (1903) On the theory of optical images, with special reference to the microscope. J R Microsc Soc 23:474–482. doi: 10.1111/j.1365-2818.1903.tb04831.x CrossRefGoogle Scholar
  122. Ries J, Kaplan C, Platonova E et al (2012) A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods 9:582–584. doi: 10.1038/nmeth.1991 PubMedCrossRefGoogle Scholar
  123. Ripley BD (1977) Modelling spatial patterns. J R Stat Soc B 39:172–212Google Scholar
  124. Ristanović Z, Kerssens MM, Kubarev AV et al (2015) High-resolution single-molecule fluorescence imaging of zeolite aggregates within real-life fluid catalytic cracking particles. Angew Chem Int Ed Engl 54:1836–1840. doi: 10.1002/anie.201410236 PubMedPubMedCentralCrossRefGoogle Scholar
  125. Ritchie K, Iino R, Fujiwara T et al (2003) The fence and picket structure of the plasma membrane of live cells as revealed by single molecule techniques (Review). Mol Membr Biol 20:13–18PubMedCrossRefGoogle Scholar
  126. Rossy J, Williamson DJ, Benzing C, Gaus K (2012) The integration of signaling and the spatial organization of the T cell synapse. Immunol Mem 3:352. doi: 10.3389/fimmu.2012.00352 Google Scholar
  127. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–796. doi: 10.1038/nmeth929 PubMedPubMedCentralCrossRefGoogle Scholar
  128. Saka SK, Honigmann A, Eggeling C et al (2014) Multi-protein assemblies underlie the mesoscale organization of the plasma membrane. Nat Commun. doi: 10.1038/ncomms5509 Google Scholar
  129. Scarselli M, Annibale P, Radenovic A (2012) Cell type-specific β2-adrenergic receptor clusters identified using photoactivated localization microscopy are not lipid raft related, but depend on actin cytoskeleton integrity. J Biol Chem 287:16768–16780. doi: 10.1074/jbc.M111.329912 PubMedPubMedCentralCrossRefGoogle Scholar
  130. Schermelleh L, Heintzmann R, Leonhardt H (2010) A guide to super-resolution fluorescence microscopy. J Cell Biol 190:165–175. doi: 10.1083/jcb.201002018 PubMedPubMedCentralCrossRefGoogle Scholar
  131. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. doi: 10.1038/nmeth.2019 PubMedCrossRefGoogle Scholar
  132. Schroeder R, London E, Brown D (1994) Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior. Proc Natl Acad Sci U S A 91:12130–12134PubMedPubMedCentralCrossRefGoogle Scholar
  133. Sengupta P, Lippincott-Schwartz J (2012) Quantitative analysis of photoactivated localization microscopy (PALM) datasets using pair-correlation analysis. BioEssays News Rev Mol Cell Dev Biol 34:396–405. doi: 10.1002/bies.201200022 CrossRefGoogle Scholar
  134. Sengupta P, Jovanovic-Talisman T, Skoko D et al (2011) Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat Methods 8:969–975. doi: 10.1038/nmeth.1704 PubMedPubMedCentralCrossRefGoogle Scholar
  135. Sengupta P, Jovanovic-Talisman T, Lippincott-Schwartz J (2013) Quantifying spatial organization in point-localization superresolution images using pair correlation analysis. Nat Protoc 8:345–354. doi: 10.1038/nprot.2013.005 PubMedPubMedCentralCrossRefGoogle Scholar
  136. Sezgin E, Gutmann T, Buhl T et al (2015) Adaptive lipid packing and bioactivity in membrane domains. PLoS One 10, e0123930. doi: 10.1371/journal.pone.0123930 PubMedPubMedCentralCrossRefGoogle Scholar
  137. Shao L, Kner P, Rego EH, Gustafsson MGL (2011) Super-resolution 3D microscopy of live whole cells using structured illumination. Nat Methods 8:1044–1046. doi: 10.1038/nmeth.1734 PubMedCrossRefGoogle Scholar
  138. Sharonov A, Hochstrasser RM (2006) Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc Natl Acad Sci 103:18911–18916. doi: 10.1073/pnas.0609643104 PubMedPubMedCentralCrossRefGoogle Scholar
  139. Sherman E, Barr V, Manley S et al (2011) Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor. Immunity 35:705–720. doi: 10.1016/j.immuni.2011.10.004 PubMedPubMedCentralCrossRefGoogle Scholar
  140. Shivanandan A, Radenovic A, Sbalzarini IF (2013) MosaicIA: an ImageJ/Fiji plugin for spatial pattern and interaction analysis. BMC Bioinformatics 14:349. doi: 10.1186/1471-2105-14-349 PubMedPubMedCentralCrossRefGoogle Scholar
  141. Shivanandan A, Unnikrishnan J, Radenovic A (2015) Accounting for limited detection efficiency and localization precision in cluster analysis in single molecule localization microscopy. PLoS One 10, e0118767. doi: 10.1371/journal.pone.0118767 PubMedPubMedCentralCrossRefGoogle Scholar
  142. Shvets E, Ludwig A, Nichols BJ (2014) News from the caves: update on the structure and function of caveolae. Curr Opin Cell Biol 29:99–106. doi: 10.1016/ PubMedCrossRefGoogle Scholar
  143. Sieber JJ, Willig KI, Kutzner C et al (2007) Anatomy and dynamics of a supramolecular membrane protein cluster. Science 317:1072–1076. doi: 10.1126/science.1141727 PubMedCrossRefGoogle Scholar
  144. Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387:569–572. doi: 10.1038/42408 PubMedCrossRefGoogle Scholar
  145. Simons K, Sampaio JL (2011) Membrane organization and lipid rafts. Cold Spring Harb Perspect Biol. doi: 10.1101/cshperspect.a004697 PubMedPubMedCentralGoogle Scholar
  146. Simons K, Toomre D (2000) Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:31–39. doi: 10.1038/35036052 PubMedCrossRefGoogle Scholar
  147. Simons K, Van Meer G (1988) Lipid sorting in epithelial cells. Biochemistry (Mosc) 27:6197–6202. doi: 10.1021/bi00417a001 CrossRefGoogle Scholar
  148. Singer SJ, Nicolson GL (1972) The fluid mosaic model of the structure of cell membranes. Science 175:720–731PubMedCrossRefGoogle Scholar
  149. Smith-Garvin JE, Koretzky GA, Jordan MS (2009) T cell activation. Annu Rev Immunol 27:591–619. doi: 10.1146/annurev.immunol.021908.132706 PubMedPubMedCentralCrossRefGoogle Scholar
  150. Solis GP, Hoegg M, Munderloh C et al (2007) Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains. Biochem J 403:313–322. doi: 10.1042/BJ20061686 PubMedPubMedCentralCrossRefGoogle Scholar
  151. Sparrow CM (1916) On spectroscopic resolving power. Astrophys J 44:76. doi: 10.1086/142271 CrossRefGoogle Scholar
  152. Steinhauer C, Forthmann C, Vogelsang J, Tinnefeld P (2008) Superresolution microscopy on the basis of engineered dark states. J Am Chem Soc 130:16840–16841. doi: 10.1021/ja806590m PubMedCrossRefGoogle Scholar
  153. Stipp CS, Kolesnikova TV, Hemler ME (2003) Functional domains in tetraspanin proteins. Trends Biochem Sci 28:106–112. doi: 10.1016/S0968-0004(02)00014-2 PubMedCrossRefGoogle Scholar
  154. Südhof TC (2012) The presynaptic active zone. Neuron 75:11–25. doi: 10.1016/j.neuron.2012.06.012 PubMedPubMedCentralCrossRefGoogle Scholar
  155. Termini CM, Cotter ML, Marjon KD et al (2014) The membrane scaffold CD82 regulates cell adhesion by altering α4 integrin stability and molecular density. Mol Biol Cell 25:1560–1573. doi: 10.1091/mbc.E13-11-0660 PubMedPubMedCentralCrossRefGoogle Scholar
  156. Tobin SJ, Cacao EE, Hong DWW et al (2014) Nanoscale effects of ethanol and naltrexone on protein organization in the plasma membrane studied by photoactivated localization microscopy (PALM). PLoS One 9, e87225. doi: 10.1371/journal.pone.0087225 PubMedPubMedCentralCrossRefGoogle Scholar
  157. Torreno-Pina JA, Castro BM, Manzo C et al (2014) Enhanced receptor–clathrin interactions induced by N-glycan–mediated membrane micropatterning. Proc Natl Acad Sci 111:11037–11042. doi: 10.1073/pnas.1402041111 PubMedPubMedCentralCrossRefGoogle Scholar
  158. Truan Z, Tarancón Díez L, Bönsch C et al (2013) Quantitative morphological analysis of arrestin2 clustering upon G protein-coupled receptor stimulation by super-resolution microscopy. J Struct Biol 184:329–334. doi: 10.1016/j.jsb.2013.09.019 PubMedCrossRefGoogle Scholar
  159. van den Bogaart G, Meyenberg K, Risselada HJ et al (2011) Membrane protein sequestering by ionic protein-lipid interactions. Nature 479:552–555. doi: 10.1038/nature10545 PubMedPubMedCentralCrossRefGoogle Scholar
  160. van Zanten TS, Cambi A, Garcia-Parajo MF (2010) A nanometer scale optical view on the compartmentalization of cell membranes. Biochim Biophys Acta BBA – Biomembr 1798:777–787. doi: 10.1016/j.bbamem.2009.09.012 CrossRefGoogle Scholar
  161. Veatch SL, Keller SL (2003) Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol. Biophys J 85:3074–3083. doi: 10.1016/S0006-3495(03)74726-2 PubMedPubMedCentralCrossRefGoogle Scholar
  162. Veatch SL, Machta BB, Shelby SA et al (2012) Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting. PLoS One. doi: 10.1371/journal.pone.0031457 PubMedPubMedCentralGoogle Scholar
  163. Whelan DR, Bell TDM (2015) Image artifacts in single molecule localization microscopy: why optimization of sample preparation protocols matters. Sci Rep. doi: 10.1038/srep07924 Google Scholar
  164. Wiegand T, Moloney KA (2013) Handbook of spatial point-pattern analysis in ecology. CRC Press, Boca RatonGoogle Scholar
  165. Williamson DJ, Owen DM, Rossy J et al (2011) Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat Immunol 12:655–662. doi: 10.1038/ni.2049 PubMedCrossRefGoogle Scholar
  166. Willig KI, Harke B, Medda R, Hell SW (2007) STED microscopy with continuous wave beams. Nat Methods 4:915–918. doi: 10.1038/nmeth1108 PubMedCrossRefGoogle Scholar
  167. Wright MD, Tomlinson MG (1994) The ins and outs of the transmembrane 4 superfamily. Immunol Today 15:588–594. doi: 10.1016/0167-5699(94)90222-4 PubMedCrossRefGoogle Scholar
  168. Xie J, Tato CM, Davis MM (2013) How the immune system talks to itself: the varied role of synapses. Immunol Rev 251:65–79. doi: 10.1111/imr.12017 PubMedPubMedCentralCrossRefGoogle Scholar
  169. Xu X, Ester M, Kriegel H-P, Sander J (1998) A distribution-based clustering algorithm for mining in large spatial databases. In: 14th international conference on data engineering, 1998. Proceedings of 14th International Conference on Data Engineering(ICDE'98), pp 324–331Google Scholar
  170. Yamada E (1955) The fine structure of the gall bladder epithelium of the mouse. J Biophys Biochem Cytol 1:445–458PubMedPubMedCentralCrossRefGoogle Scholar
  171. Yáñez-Mó M, Barreiro O, Gordon-Alonso M et al (2009) Tetraspanin-enriched microdomains: a functional unit in cell plasma membranes. Trends Cell Biol 19:434–446. doi: 10.1016/j.tcb.2009.06.004 PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Laura Paparelli
    • 1
    • 2
    • 3
  • Nikky Corthout
    • 1
    • 4
    • 5
  • Benjamin Pavie
    • 1
    • 4
  • Wim Annaert
    • 2
    • 3
  • Sebastian Munck
    • 1
    • 4
    • 5
    Email author
  1. 1.VIB Bio Imaging CoreLeuvenBelgium
  2. 2.Laboratory of Membrane Trafficking, Department of Human GeneticsKU LeuvenLeuvenBelgium
  3. 3.VIB Center for the Biology of DiseaseKU LeuvenLeuvenBelgium
  4. 4.Department of Human GeneticsVIB Center for the Biology of Disease, KU LeuvenLeuvenBelgium
  5. 5.VIB, LiMoNeLeuvenBelgium

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