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

Abstract

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.

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain

“…of energy homotransfer (energy migration) ( 35 ). The effi ciency of energy transfer will increase as the distance that separates the two chromophores decreases.…”

Detection of Lipid Domains in Model and Cell Membranes by Fluorescence Lifetime Imaging Microscopy of Fluorescent Lipid Analogues

“…Using headgroup-labeled Rhodamine dioleoylphosphatidylethanolamine (N-Rho-DOPE) de Almeida et al (27) could distinguish three lipid phases, gel, lo, and ld, in GUVs by fluorescence lifetime imaging microscopy (FLIM). Margineanu et al (28) have successfully applied a peryleneimide probe to visualize the lo and the ld phase in GUVs by fluorescence intensity and FLIM.…”

Optical Characterization and Lasing in Three-Dimensional Opal-Structures

“…Studies of energy transfer in combinations of pyrene-perylene, two different coumarine dyes, and fluorescein-ethidium bromide, have been carried out with applications in biological imaging and bioanalytical science (Takakusa et al, 2003;Fujiwara et al, 2005;de Almeida et al, 2007;Stevens et al, 2009). Overlap of the fluorescence and absorption was estimated from our experiments as 6.9 × 10 −15 mol −1 cm 6 (Figure 8), and the critical Förster radius was calculated as 3.1 nm.…”