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Biophys J. 2013 Jul 2;105(1):127-36. doi: 10.1016/j.bpj.2013.05.043.

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

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  • 1Aix Marseille Université, CNRS, Centrale Marseille, Institut Fresnel, UMR 7249, F-13013 Marseille, France.

Abstract

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization.

Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

PMID:
23823231
[PubMed - indexed for MEDLINE]
PMCID:
PMC3699755
Free PMC Article
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