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Improved Characterization of Raft-Mimicking Phase-Separation Phenomena in Lipid Bilayers Using Laurdan Fluorescence

Nicolas Puff1,2, Galya Staneva3, Miglena I Angelova1,2

  • 1Sorbonne Université, Faculté des Sciences et Ingénierie, UFR 925 Physique, Paris F-75005, France.

Langmuir : the ACS Journal of Surfaces and Colloids
|April 3, 2020
PubMed
Summary

This study enhances spectral decomposition of Laurdan fluorescence to better analyze liquid-ordered (Lo)-liquid-disordered (Ld) phase separation in model membranes. A new ratio (Rx) offers a sensitive index for phase separation, improving understanding of membrane properties.

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Area of Science:

  • Biophysics
  • Membrane Biophysics
  • Lipid Bilayer Systems

Background:

  • Biomimetic model membrane systems are crucial for understanding lipid rafts in plasma membranes.
  • Laurdan fluorescence probe and general polarization (GP) parameter are used to study liquid-ordered (Lo)-liquid-disordered (Ld) phase separation.
  • GP parameter provides global average packing but cannot determine individual phase contributions.

Purpose of the Study:

  • To develop and validate a spectral decomposition method for Laurdan fluorescence to overcome GP limitations in studying Lo-Ld phase separation.
  • To investigate Laurdan in sphingomyelin-cholesterol-phosphatidylcholine vesicles undergoing phase separation.
  • To establish a new index for quantifying Lo-Ld phase separation and its sensitivity compared to GP.

Main Methods:

  • Utilized spectroscopic methods, specifically Laurdan fluorescence emission spectra decomposition into log-normal components.
  • Investigated lipid compositions for homogeneous Lo/Ld phases and thermally induced Lo-Ld phase separation.
  • Employed fluorescence imaging of giant unilamellar vesicles to confirm phase separation.
  • Analyzed the ratio Rx (intermediate-energy peak to low-energy peak) as an index for phase separation.

Main Results:

  • Decomposition into three log-normal components revealed an intermediate energy component specific to the Lo phase.
  • A small contribution of the intermediate component above phase separation temperature indicates phase fluctuations.
  • The ratio Rx proved to be a sensitive index for Lo-Ld phase separation, sometimes exceeding GP sensitivity.
  • Rx combined with GP provided deeper insights into phase separation, exemplified by butanol's effect on line tension.

Conclusions:

  • The three-component log-normal decomposition of Laurdan spectra offers an improved method for characterizing Lo-Ld phase-separation phenomena.
  • The Rx ratio provides a straightforward and sensitive index for quantifying Lo-Ld phase separation.
  • This enhanced spectral analysis, combined with GP, offers a more comprehensive understanding of lipid raft behavior and membrane dynamics.