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Complexity of Lipid Domains and Rafts in Giant Unilamellar Vesicles Revealed by Combining Imaging and Microscopic and Macroscopic Time-Resolved Fluorescence

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

^* MicroSpectroscopy Centre, Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands; Centro de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal; and Centro de Química-Física Molecular, Instituto Superior Técnico da Universidade Técnica de Lisboa, Lisbon, Portugal

Correspondence: Address reprint requests to Rodrigo F. M. de Almeida, E-mail: r.almeida{at}mail.ist.utl.pt.

The application of fluorescence lifetime imaging microscopy to study gel/fluid and raftlike lipid domains in giant unilamellar vesicles (GUVs) is demonstrated here. Different regions of the ternary dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine/cholesterol phase diagram were studied. The head-labeled phospholipid Rhodamine-dioleoylphosphatidylethanolamine (Rhod-DOPE) was used as a fluorescent probe. Gel/fluid and liquid-ordered (l_o)/liquid-disordered (l_d) 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 nongiant vesicle suspensions. The fingerprints for gel/fluid coexistence and for the presence of l_o raftlike phase, based on fluorescence lifetime imaging microscopy histograms and images, and on the fluorescence intensity decay parameters of Rhod-DOPE, are presented. The presence of three lipid phases in one single GUV is detected unequivocally. From the comparison of lifetime parameters, it can be concluded that the l_o phase is formed in the binary dipalmitoylphosphatidylcholine/cholesterol but not in the dioleoylphosphatidylcholine/cholesterol mixture. The domains apparent in fluorescence intensity images have a more complex substructure revealed by analysis of the lifetime data. The potential applications of this combined imaging/microscopic/macroscopic methodology are discussed.

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