Skip to main content

Imaging Membrane Order Using Environmentally Sensitive Fluorophores

  • Protocol
  • First Online:
Methods in Membrane Lipids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1232))

Abstract

In the lipid raft hypothesis, ordered and disordered lipid membranes are responsible for regulating the distribution, dynamics, and interactions of membrane associated proteins. Ordered and disordered bilayers may be distinguished by the degree of order in their acyl tails (the order parameter) which in turn affects lipid mobility and lipid packing. Low density lipid packing in the disordered phase allows polar water molecules to penetrate into the usually non-polar bilayer interior. Transition to the ordered phase causes condensation of the membrane, tighter lipid packing, and more complete exclusion of polar water. This process can be measured and quantified using polarity sensitive fluorophores embedded within the bilayer which then have different emission properties depending on membrane phase. Two examples of these are Laurdan and di-4-ANEPPDHQ which can be used to image membrane order distributions in live cells via a variety of microscopy techniques.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387:569–572

    Article  PubMed  CAS  Google Scholar 

  2. Lingwood D, Simons K (2010) Lipid rafts as a membrane-organizing principle. Science 327(5961):46–50

    Article  PubMed  CAS  Google Scholar 

  3. Gaus K et al (2005) Condensation of the plasma membrane at the site of T lymphocyte activation. J Cell Biol 171(1): 121–131

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  4. Owen DM et al (2010) High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells. Mol Membr Biol 27(4–6):178–189

    Article  PubMed  CAS  Google Scholar 

  5. van Meer G et al (1987) Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells. J Cell Biol 105(4):1623–1635

    Article  PubMed  Google Scholar 

  6. Carter GC et al (2009) HIV entry in macrophages is dependent on intact lipid rafts. Virology 386(1):192–202

    Article  PubMed  CAS  Google Scholar 

  7. Takeda M et al (2003) Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion. Proc Natl Acad Sci 100(25):14610–14617

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  8. Rentero C et al (2008) Functional implications of plasma membrane condensation for T cell activation. PLoS One 3(5):e2262

    Article  PubMed  PubMed Central  Google Scholar 

  9. Williamson DJ et al (2011) Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat Immunol 12(7):655–662

    Article  PubMed  CAS  Google Scholar 

  10. Gaus K et al (2006) Integrin-mediated adhesion regulates membrane order. J Cell Biol 174(5):725–734

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  11. Khurana S et al (2007) Human immunodeficiency virus type 1 and influenza virus exit via different membrane microdomains. J Virol 81(22):12630–12640

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  12. Carrasco M, Amorim MJ, Digard P (2004) Lipid raft-dependent targeting of the influenza A virus nucleoprotein to the apical plasma membrane. Traffic 5(12):979–992

    Article  PubMed  CAS  Google Scholar 

  13. Lagerholm BC et al (2005) Detecting microdomains in intact cell membranes. Annu Rev Phys Chem 56:309–336

    Article  PubMed  CAS  Google Scholar 

  14. Owen DM et al (2007) Optical techniques for imaging membrane lipid microdomains in living cells. Semin Cell Dev Biol 18(5):591–598

    Article  PubMed  CAS  Google Scholar 

  15. Parasassi T et al (1998) Laurdan and Prodan as polarity-sensitive fluorescent membrane probes. J Fluoresc 8(4):365–373

    Article  CAS  Google Scholar 

  16. Parasassi T et al (1991) Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence. Biophys J 60(1):179–189

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  17. Jin L et al (2005) Cholesterol-enriched lipid domains can be visualized by di-4-ANEPPDHQ with linear and nonlinear optics. Biophys J 89(1):L04–L06

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  18. Owen DM et al (2006) Fluorescence lifetime imaging provides enhanced contrast when imaging the phase-sensitive dye di-4-ANEPPDHQ in model membranes and live cells. Biophys J 90(11):L80–L82

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  19. Kwiatek JM et al (2013) Characterization of a new series of fluorescent probes for imaging membrane order. PLoS One 8(2):e52960

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  20. Owen DM et al (2012) Sub-resolution lipid domains exist in the plasma membrane and regulate protein diffusion and distribution. Nat Commun 3:1256

    Article  PubMed  Google Scholar 

  21. Golfetto O, Hinde E, Gratton E (2013) Laurdan fluorescence lifetime discriminates cholesterol content from changes in fluidity in living cell membranes. Biophys J 104(6):1238–1247

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  22. Owen DM et al (2011) Quantitative imaging of membrane lipid order in cells and organisms.Nat Protoc 7(1): 24–35

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dylan M. Owen .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Ashdown, G.W., Owen, D.M. (2015). Imaging Membrane Order Using Environmentally Sensitive Fluorophores. In: Owen, D. (eds) Methods in Membrane Lipids. Methods in Molecular Biology, vol 1232. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1752-5_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1752-5_10

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1751-8

  • Online ISBN: 978-1-4939-1752-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics