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Biophys. J. BioFAST: First Published January 13, 2006. doi:10.1529/biophysj.105.072884
© 2006 by the Biophysical Society.

A more recent version of this article appeared on April 1, 2006.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Characterization and Application of a New Optical Probe for Membrane Lipid Domains

Lei Jin 1, Andrew C. Millard 1, Joseph P. Wuskell 1, Xuemei Dong 2, Dianqing Wu 2, Heather A. Clark 1 and Leslie M. Loew 1*

1 Center for Cell Analysis and Modeling,University of Connecticut Health Center
2 Department of Immunology, University of Connecticut Health Center

* To whom correspondence should be addressed. E-mail: les{at}volt.uchc.edu.

Submitted on August 18, 2005
Revised on September 26, 2005
Accepted on 15 December 2005


  Abstract
In this paper, we characterize the fluorescence of an environmentally sensitive probe for lipid membranes, di-4-ANEPPDHQ. In large unilamellar lipid vesicles (LUV), its emission spectrum shifts up to 30nm to the blue with increasing cholesterol concentration. Independently, it displays a comparable blue shift in liquid-ordered relative to liquid-disordered phases. The cumulative effect is a 60nm difference in emission spectra for cholesterol containing LUVs in the liquid ordered state vs. cholesterol-free LUVs in the liquid disordered phase. Given these optical properties, we use di-4-ANEPPDHQ to image the phase separation in giant unilamellar vesicles with both linear and non-linear optical microscopies. The dye shows green and red fluorescence in liquid-ordered and disordered domains, respectively. We propose that this reflects the relative rigidity of the molecular packing around the dye molecules in the two phases. We also observe a 7-fold stronger second harmonic generation signal in the liquid-disordered domains, consistent with a higher concentration of the dye resulting from preferential partitioning into the disordered phase. The efficacy of the dye to report lipid domains in cell membranes is demonstrated in polarized migrating neutrophils.

Key Words: cholesterol, di-4-ANEPPDHQ, fluorescence, membrane, phase, second harmonic generation




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Copyright © 2006 by the Biophysical Society.