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Biophys. J. BioFAST: First Published April 20, 2007. doi:10.1529/biophysj.106.098822
© 2007 by the Biophysical Society.

A more recent version of this article appeared on July 15, 2007.
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MEMBRANES

Complexity of lipid domains and rafts in giant unilamellar vesicles revealed by combining imaging, microscopic and macroscopic time-resolved fluorescence

Rodrigo F. M. de Almeida 1*, JanWillem Borst 2, Alexander Fedorov 1, Manuel Prieto 1 and Antonie J. W. G. Visser 2

1 Instituto Superior Técnico
2 Wageningen University

* To whom correspondence should be addressed. E-mail: r.almeida{at}mail.ist.utl.pt.

Submitted on October 3, 2006
Revised on November 29, 2006
Accepted on 21 March 2007


  Abstract
The application of fluorescence lifetime imaging microscopy (FLIM) to study gel/fluid and raft-like lipid domains in giant unilamellar vesicles (GUV) is demonstrated here. Different regions of the ternary dipalmitoylphosphatidylcholine (DPPC)/ dioleoylphosphatidylcholine (DOPC)/ cholesterol phase diagram were studied. The head-labeled phospholipid Rhodamine-dioleoylphosphatidylethanolamine (Rhod-DOPE) was used as a fluorescent probe. Gel/fluid and liquid ordered (lo)/liquid disordered (ld) phase separation were clearly visualized upon two-photon excitation. Fluorescence intensity decays in different regions of a GUV were also obtained with the microscope in fixed laser beam configuration. The ensemble behavior of the system was studied by obtaining fluorescence intensity decays of Rhod-DOPE in non-giant vesicle suspensions. The fingerprints for gel/fluid coexistence and for the presence of lo raft-like phase, based on FLIM histograms and images, and on the fluorescence intensity decay parameters of Rhod-DOPE, are presented. The presence of 3 lipid phases in one single GUV is detected unequivocally. From the comparison of lifetime parameters, it can be concluded that the lo phase is formed in the binary DPPC/cholesterol but not in the DOPC/cholesterol mixture. The domains apparent in fluorescence intensity images have a more complex sub-structure revealed by analysis of the lifetime data. The potential applications of this combined imaging/microscopic/macroscopic methodology are discussed.

Key Words: FLIM, giant liposomes, lipid phase separation, lipid rafts, time-resolved fluorescence spectroscopy, two-photon microscopy




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Copyright © 2007 by the Biophysical Society.