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Characterization of fluorescent probe partitioning in giant unilamellar vesicles of “lipid raft” mixtures using confocal fluorescence microscopy
Author(s) -
Sharom Frances J.,
Juhasz Janos,
Davis James H.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.689.3
Subject(s) - fluorescence , vesicle , lipid raft , bodipy , raft , lipid microdomain , lipid bilayer , chemistry , fluorescence microscope , phase (matter) , microscopy , popc , biophysics , förster resonance energy transfer , confocal microscopy , rhodamine , membrane , biology , polymer , biochemistry , organic chemistry , optics , physics , copolymer , microbiology and biotechnology
Direct visualization of raft‐like liquid ordered (l o ) domains requires fluorescence probes with known partitioning preference for a specific lipid phase. Therefore, a detailed understanding of the behaviour of commonly used fluorescent probes in defined lipid bilayer systems is needed before they can be used to draw conclusions about the physical state of the membrane. Using giant unilamellar vesicles composed of a ternary “lipid raft” mixture (DOPC/DPPC/cholesterol) for which the phase behaviour is known, we examined nine commonly used fluorescent probes using confocal fluorescence microscopy. The partitioning preference of each probe was assigned using either a well‐characterized l d phase marker, or by quantitation of the domain area fraction. Fluorescent molecules were examined individually, in pairs, and in threes; most of them partitioned into the l d phase, while two probes preferred the l o phase or gel phase. Interestingly, the partitioning of DiIC 18 was influenced by Bodipy‐PC, and Lissamine rhodamine B‐DPPE affected the partitioning preference of other fluorescence probes when used with them. We compare and contrast the different fluorescence probes in terms of their partitioning preference, ability to detect phase separations, photostability, and any induced change in lipid miscibility transition temperature. Supported by the Natural Sciences and Engineering Research Council of Canada