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Biophys J, October 1999, p. 2266-2283, Vol. 77, No. 4
*Biophysics Research Division and Department of Physics, and #Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109 USA
In living cells, variations in membrane orientation occur
both in easily imaged large-scale morphological features, and also in
less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method is introduced here to visualize such regions. The
method is based on fluorescence of an oriented membrane probe excited
by a polarized evanescent field created by total internal reflection
(TIR) illumination. The fluorescent carbocyanine dye diI-C18-(3) (diI) has previously been shown to embed in the
lipid bilayer of cell membranes with its transition dipoles oriented nearly in the plane of the membrane. The membrane-embedded diI near the
cell-substrate interface can be fluorescently excited by evanescent
field light polarized either perpendicular or parallel to the plane of
the substrate coverslip. The excitation efficiency from each
polarization depends on the membrane orientation, and thus the ratio of
the observed fluorescence excited by these two polarizations vividly
shows regions of microscopic and submicroscopic curvature of the
membrane, and also gives information regarding the fraction of
unoriented diI in the membrane. Both a theoretical background and
experimental verification of the technique is presented for samples of
1) oriented diI in model lipid bilayer membranes, erythrocytes, and
macrophages; and 2) randomly oriented fluorophores in rhodamine-labeled
serum albumin adsorbed to glass, in rhodamine dextran solution, and in
rhodamine dextran-loaded macrophages. Sequential digital images of the
polarized TIR fluorescence ratios show spatially-resolved time-course
maps of membrane orientations on diI-labeled macrophages from which low
visibility membrane structures can be identified and quantified. To
sharpen and contrast-enhance the TIR images, we deconvoluted them with
an experimentally measured point spread function. Image deconvolution
is especially effective and fast in our application because
fluorescence in TIR emanates from a single focal plane.
Biophys J, October 1999, p. 2266-2283, Vol. 77, No. 4
© 1999 by the Biophysical Society 0006-3495/99/10/2266/18 $2.00
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