Skip to main content
SpringerLink
Log in

Investigating the Dynamics and Organization of Membrane Proteins and Lipids by Imaging Fluorescence Correlation Spectroscopy

  • Chapter
  • First Online:
Membrane Organization and Dynamics

Part of the book series: Springer Series in Biophysics ((BIOPHYSICS,volume 20))

Abstract

The dynamics and organization of lipid bilayers, whether they are artificial supported lipid bilayers, lipid vesicles or cell membranes, still pose an enigma. Especially bilayers with multiple lipid components, not to mention peptides and proteins, are difficult to characterize as they often exhibit fast molecular dynamics and structural organization that presumably are on the nanometer scale. Therefore, biophysical techniques are required that measure sufficiently fast to detect molecular movements and interactions but also provide information about structures below the optical diffraction limit. Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) fulfils these conditions and can resolve membrane dynamics with high temporal resolution and provide information even on nanoscopic scales. Compared to conventional confocal FCS, this multiplexed modality can record over hundreds of contiguous points simultaneously on the membrane. In this chapter, we present briefly the theory of Imaging FCS and provide general guidelines for its implementation. This is followed by a description of multiple options to analyse the Imaging FCS data. We discuss the FCS diffusion law to investigate the membrane organization below the optical diffraction limit, the difference in cross-correlation function (ΔCCF) to investigate anisotropies in diffusion, Imaging Fluorescence Cross-correlation (Imaging FCCS) to study interactions, and the recovery of the Arrhenius activation energy of diffusion to determine lipid packing and phases. Lastly, we give a short overview of recent applications of Imaging FCS.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Garcia-Parajo MF, Cambi A, Torreno-Pina JA, Thompson N, Jacobson K. Nanoclustering as a dominant feature of plasma membrane organization. J Cell Sci. 2014;127:4995–5005.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Raghupathy R, Anilkumar AA, Polley A, Singh PP, Yadav M, Johnson C, Suryawanshi S, Saikam V, Sawant SD, Panda A, Guo Z, Vishwakarma RA, Rao M, Mayor S. Transbilayer lipid interactions mediate nanoclustering of lipid-anchored proteins. Cell. 2015;161:581–94.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Rao M, Mayor S. Active organization of membrane constituents in living cells. Curr Opin Cell Biol. 2014;29:126–32.

    Article  PubMed  CAS  Google Scholar 

  4. Lingwood D, Simons K. Lipid rafts as a membrane-organizing principle. Science. 2010;327:46–50.

    Article  PubMed  CAS  Google Scholar 

  5. Nicolson GL. Fluid-mosaic membrane structure: from cellular control and domains to extracellular vesicles. In: Membrane organization and lipid rafts in cells and artificial membranes. New York: Nova Science Publishers; 2016. p. 1–23.

    Google Scholar 

  6. Kusumi A, Fujiwara TK, Chadda R, Xie M, Tsunoyama TA, Kalay Z, Kasai RS, Suzuki KG. Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson’s fluid-mosaic model. Annu Rev Cell Dev Biol. 2012;28:215–50.

    Article  PubMed  CAS  Google Scholar 

  7. Kraft ML. Plasma membrane organization and function: moving past lipid rafts. Mol Biol Cell. 2013;24:2765–8.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Kusumi A, Suzuki KG, Kasai RS, Ritchie K, Fujiwara TK. Hierarchical mesoscale domain organization of the plasma membrane. Trends Biochem Sci. 2011;36:604–15.

    Article  PubMed  CAS  Google Scholar 

  9. Truong-Quang BA, Lenne PF. Membrane microdomains: from seeing to understanding. Front Plant Sci. 2014;5:18.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Yap AS, Gomez GA, Parton RG. Adherens junctions revisualized: organizing cadherins as nanoassemblies. Dev Cell. 2015;35:12–20.

    Article  PubMed  CAS  Google Scholar 

  11. Suzuki KG, Kasai RS, Hirosawa KM, Nemoto YL, Ishibashi M, Miwa Y, Fujiwara TK, Kusumi A. Transient GPI-anchored protein homodimers are units for raft organization and function. Nat Chem Biol. 2012;8:774–83.

    Article  PubMed  CAS  Google Scholar 

  12. Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol. 2000;1:31–9.

    Article  PubMed  CAS  Google Scholar 

  13. van Zanten TS, Mayor S. Current approaches to studying membrane organization. F1000Research; 2015.

    Google Scholar 

  14. Manzo C, Garcia-Parajo MF. A review of progress in single particle tracking: from methods to biophysical insights. Rep Prog Phys. 2015;78:124601.

    Article  PubMed  CAS  Google Scholar 

  15. Manley S, Gillette JM, Patterson GH, Shroff H, Hess HF, Betzig E, Lippincott-Schwartz J. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods. 2008;5:155–7.

    Article  PubMed  CAS  Google Scholar 

  16. Serge A, Bertaux N, Rigneault H, Marguet D. Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat Methods. 2008;5:687–94.

    Article  PubMed  CAS  Google Scholar 

  17. Giannone G, Hosy E, Levet F, Constals A, Schulze K, Sobolevsky AI, Rosconi MP, Gouaux E, Tampe R, Choquet D, Cognet L. Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys J. 2010;99:1303–10.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Moertelmaier M, Brameshuber M, Linimeier M, Schütz GJ, Stockinger H. Thinning out clusters while conserving stoichiometry of labeling. Appl Phys Lett. 2005;87:263903.

    Article  CAS  Google Scholar 

  19. Ries J, Weidemann T, Schwille P. Fluorescence correlation spectroscopy. In: Egelman EH, editor. Comprehensive biophysics. Amsterdam: Elsevier; 2012. p. 210–45.

    Chapter  Google Scholar 

  20. Kannan B, Guo L, Sudhaharan T, Ahmed S, Maruyama I, Wohland T. Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera. Anal Chem. 2007;79:4463–70.

    Article  PubMed  CAS  Google Scholar 

  21. Petrov EP, Schwille P. State of the art and novel trends in fluorescence correlation spectroscopy. Springer Ser Fluoresc. 2008;6:145–97.

    Article  CAS  Google Scholar 

  22. Sankaran J, Bag N, Kraut RS, Wohland T. Accuracy and precision in camera-based fluorescence correlation spectroscopy measurements. Anal Chem. 2013;85:3948–54.

    Article  PubMed  CAS  Google Scholar 

  23. Zhang B, Zerubia J, Olivo-Marin JC. A study of Gaussian approximations of fluorescence microscopy PSF models. Proceedings of SPIE 6090, Three-dimensional and multidimensional microscopy: image acquisition and processing XIII 6090:60900K-60900K-60911; 2006.

    Google Scholar 

  24. Bag N, Sankaran J, Paul A, Kraut RS, Wohland T. Calibration and limits of camera-based fluorescence correlation spectroscopy: a supported lipid bilayer study. Chem Phys Chem. 2012;13:2784–94.

    Article  PubMed  CAS  Google Scholar 

  25. Bag N, Wohland T. Imaging fluorescence fluctuation spectroscopy: new tools for quantitative bioimaging. Annu Rev Phys Chem. 2014;65:225–48.

    Article  PubMed  CAS  Google Scholar 

  26. Singh AP, Wohland T. Applications of imaging fluorescence correlation spectroscopy. Curr Opin Chem Biol. 2014;20:29–35.

    Article  PubMed  CAS  Google Scholar 

  27. Axelrod D. Total internal reflection fluorescence microscopy in cell biology. Traffic. 2001;2:764–74.

    Article  PubMed  CAS  Google Scholar 

  28. Krieger JW, Singh AP, Bag N, Garbe CS, Saunders TE, Langowski J, Wohland T. Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms. Nat Protoc. 2015;10:1948–74.

    Article  PubMed  CAS  Google Scholar 

  29. Sankaran J, Manna M, Guo L, Kraut R, Wohland T. Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy. Biophys J. 2009;97:2630–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Singh AP, Krieger JW, Buchholz J, Charbon E, Langowski J, Wohland T. The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy. Opt Express. 2013;21:8652–68.

    Article  PubMed  CAS  Google Scholar 

  31. Burkhardt M, Schwille P. Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy. Opt Express. 2006;14:5013–20.

    Article  PubMed  Google Scholar 

  32. Guo SM, Bag N, Mishra A, Wohland T, Bathe M. Bayesian total internal reflection fluorescence correlation spectroscopy reveals hIAPP-induced plasma membrane domain organization in live cells. Biophys J. 2014;106:190–200.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Hirsch M, Wareham RJ, Martin-Fernandez ML, Hobson MP, Rolfe DJ. A stochastic model for electron multiplication charge-coupled devices—from theory to practice. PLoS One. 2013;8:e53671.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Zhang B, Zerubia J, Olivo-Marin JC. Gaussian approximations of fluorescence microscope point-spread function models. Appl Opt. 2007;46:1819–29.

    Article  PubMed  Google Scholar 

  35. Petrasek Z, Schwille P. Photobleaching in two-photon scanning fluorescence correlation spectroscopy. Chemphyschem Eur J Chem Phys Phys Chem. 2008;9:147–58.

    Article  CAS  Google Scholar 

  36. Ries J, Chiantia S, Schwille P. Accurate determination of membrane dynamics with line-scan FCS. Biophys J. 2009;96:1999–2008.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Macháň R, Foo YH, Wohland T. On the equivalence of FCS and FRAP: simultaneous lipid membrane measurements. Biophys J. 2016;111(1):152–61.

    Google Scholar 

  38. Meseth U, Wohland T, Rigler R, Vogel H. Resolution of fluorescence correlation measurements. Biophys J. 1999;76:1619–31.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Wohland T, Rigler R, Vogel H. The standard deviation in fluorescence correlation spectroscopy. Biophys J. 2001;80:2987–99.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Saffarian S, Elson EL. Statistical analysis of fl uorescence correlation spectroscopy: the standard Deviation and bias. Biophys J. 2003;84:2030–42.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Schatzel K, Peters R. Noise on multiple-tau photon-correlation data. Proc SPIE Photon Correl Spectrosc Multicomponent Syst. 1991;1430:109–15.

    Article  Google Scholar 

  42. Sengupta P, Garai K, Balaji J, Periasamy N, Maiti S. Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy. Biophys J. 2003;84:1977–84.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Modos K, Galantai R, Bardos-Nagy I, Wachsmuth M, Toth K, Fidy J, Langowski J. Maximum-entropy decomposition of fluorescence correlation spectroscopy data: application to liposome-human serum albumin association. Eur Biophys J. 2004;33:59–67.

    Article  PubMed  CAS  Google Scholar 

  44. He J, Guo SM, Bathe M. Bayesian approach to the analysis of fluorescence correlation spectroscopy data I: theory. Anal Chem. 2012;84:3871–9.

    Article  PubMed  CAS  Google Scholar 

  45. Guo SM, He J, Monnier N, Sun G, Wohland T, Bathe M. Bayesian approach to the analysis of fluorescence correlation spectroscopy data II: application to simulated and in vitro data. Anal Chem. 2012;84:3880–8.

    Article  PubMed  CAS  Google Scholar 

  46. Wawrezinieck L, Rigneault H, Marguet D, Lenne P. Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization. Biophys J. 2005;89:4029–42.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Ng XW, Bag N, Wohland T. Characterization of lipid and cell membrane organization by the fluorescence correlation spectroscopy diffusion law. CHIMIA Int J Chem. 2015;69:112–9.

    Article  CAS  Google Scholar 

  48. Bag N, Ng XW, Sankaran J, and Wohland T. Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes. Methods Appl. in Fluoresc. 2016;4(3):034003.

    Google Scholar 

  49. Di Rienzo C, Gratton E, Beltram F, Cardarelli F. Fast spatiotemporal correlation spectroscopy to determine protein lateral diffusion laws in live cell membranes. Proc Natl Acad Sci U S A. 2013;110:12307–12.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Cardarelli F, Gratton E. Spatiotemporal fluorescence correlation spectroscopy of inert tracers: a journey within cells, one molecule at a time. Berlin: Springer; 2016. p. 1–23.

    Google Scholar 

  51. Moens PD, Digman MA, Gratton E. Modes of diffusion of cholera toxin bound to GM1 on live cell membrane by image mean square displacement analysis. Biophys J. 2015;108:1448–58.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Bag N, Ali A, Chauhan VS, Wohland T, Mishra A. Membrane destabilization by monomeric hIAPP observed by imaging fluorescence correlation spectroscopy. Chem Commun (Camb). 2013;49:9155–7.

    Article  CAS  Google Scholar 

  53. Bag N, Huang S, Wohland T. Plasma membrane organization of epidermal growth factor receptor in resting and ligand-bound states. Biophys J. 2015;109:1925–36.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Huang H, Simsek MF, Jin W, Pralle A. Effect of receptor dimerization on membrane lipid raft structure continuously quantified on single cells by camera based fluorescence correlation spectroscopy. PLoS One. 2015;10:e0121777.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Kohl T, Haustein E, Schwille P. Determining protease activity in vivo by fluorescence cross-correlation analysis. Biophys J. 2005;89:2770–82.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Unruh JR, Gratton E. Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera. Biophys J. 2008;95:5385–98.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Guo L, Har JY, Sankaran J, Hong Y, Kannan B, Wohland T. Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study. Chemphyschem Eur J Chem Phys Phys Chem. 2008;9:721–8.

    Article  CAS  Google Scholar 

  58. Ng XW, Teh C, Korzh V, Wohland T. The secreted signaling protein wnt3 is associated with membrane domains in vivo: a SPIM-FCS study. Biophys J. 2016;111(2):418–29.

    Google Scholar 

  59. Bag N, Yap DHX, Wohland T. Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy. Biochim Biophys Acta Biomembr. 2014;1838:802–13.

    Article  CAS  Google Scholar 

  60. Sankaran J, Shi X, Ho LY, Stelzer EH, Wohland T. ImFCS: a software for imaging FCS data analysis and visualization. Opt Express. 2010;18:25468–81.

    Article  PubMed  Google Scholar 

  61. Arndt-Jovin DJ, Botelho MG, Jovin TM. Structure-function relationships of ErbB RTKs in the plasma membrane of living cells. Cold Spring Harb Perspect Biol. 2014;6:a008961.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Sigismund S, Argenzio E, Tosoni D, Cavallaro E, Polo S, Di Fiore PP. Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation. Dev Cell. 2008;15:209–19.

    Article  PubMed  CAS  Google Scholar 

  63. Struntz P, Weiss M. Multiplexed measurement of protein diffusion in Caenorhabditis elegans embryos with SPIM-FCS. J Phys D Appl Phys. 2016;49:044002.

    Article  CAS  Google Scholar 

  64. Kisley L, Brunetti R, Tauzin LJ, Shuang B, Yi X, Kirkeminde AW, Higgins DA, Weiss S, Landes CF. Characterization of porous materials by fluorescence correlation spectroscopy super-resolution optical fluctuation imaging. ACS Nano. 2015;9:9158–66.

    Article  PubMed  CAS  Google Scholar 

  65. Ashdown GW, Cope A, Wiseman PW, Owen DM. Molecular flow quantified beyond the diffraction limit by spatiotemporal image correlation of structured illumination microscopy data. Biophys J. 2014;107:L21–3.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Xue Wen Ng for help with graphics and Jagadish Sankaran for critical comments on the manuscript. T.W. gratefully acknowledges the funding by the Ministry of Education Singapore (MOE2012-T3-1-008). N.B. was the recipient of a postdoctoral fellowship from the same grant. S.H. is the recipient of a graduate scholarship of the National University of Singapore.

Author information

Authors and Affiliations

  1. Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117557, Singapore

    Nirmalya Bag, Shuangru Huang & Thorsten Wohland

  2. Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore, 117543, Singapore

    Thorsten Wohland

Authors
  1. Nirmalya Bag
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuangru Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thorsten Wohland
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thorsten Wohland .

Editor information

Editors and Affiliations

  1. Centre for Cellular & Molecular Biology , Hyderabad, India

    Amitabha Chattopadhyay

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer International Publishing AG

About this chapter

Check for updates. Verify currency and authenticity via CrossMark

Cite this chapter

Bag, N., Huang, S., Wohland, T. (2017). Investigating the Dynamics and Organization of Membrane Proteins and Lipids by Imaging Fluorescence Correlation Spectroscopy. In: Chattopadhyay, A. (eds) Membrane Organization and Dynamics . Springer Series in Biophysics, vol 20. Springer, Cham. https://doi.org/10.1007/978-3-319-66601-3_6

Download citation

Publish with us

Policies and ethics

Search

Navigation