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Biophys. J. BioFAST: First Published November 1, 2004. doi:10.1529/biophysj.104.050096
© 2004 by the Biophysical Society.

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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Fluorescence Imaging of Two-Photon Linear Dichroism: Cholesterol Depletion disrupts Molecular Orientation in Cell Membranes

Richard K P Benninger 1, Bjorn Onfelt 1*, Mark AA Neil 1, Daniel M Davis 1 and Paul MW French 1

1 Imperial College London

* To whom correspondence should be addressed. E-mail: b.onfelt{at}imperial.ac.uk.

Submitted on July 24, 2004
Revised on August 12, 2004
Accepted on 19 October 2004


  Abstract
The plasma membrane of cells is an ordered environment, giving rise to anisotropic orientation and restricted motion of molecules and proteins residing in the membrane. At the same time as being an organized matrix of defined structure, the cell membrane is heterogeneous and dynamic. Here we present a method where we use fluorescence imaging of linear dichroism (LD) to measure the steady-state orientation of molecules relative to the cell membrane. By detecting LD as well as fluorescence anisotropy, the steady-state orientation parameters are separated from dynamic properties such as rotational diffusion and homo energy transfer (energy migration). The sensitivity of the technique is enhanced by using two-photon excitation for higher photo-selection compared to single photon excitation. We show here that we can accurately image lipid organization in whole cell membranes and in delicate structures such as membrane nanotubes connecting two cells. The speed of our wide-field imaging system makes it possible to image changes in orientation and anisotropy occuring on a sub-second time scale. This is demonstrated by time-lapse studies showing that cholesterol depletion rapidly disrupts the orientation of a fluorophore located within the hydrophobic region of the cell membrane but not of a surface bound probe. This is consistent with cholesterol having an important role in stabilizing and ordering the lipid tails within the plasma membrane.

Key Words: Fluorescence imaging, Linear dichroism, anisotropy, cell membrane, microscopy, multiphoton




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Copyright © 2004 by the Biophysical Society.