In living cells, variations in membrane orientation occur both in easily im
aged large-scale morphological features, and also in less visualizable subm
icroscopic regions of activity such as endocytosis, exocytosis, and cell su
rface ruffling. A fluorescence microscopic method is introduced here to vis
ualize such regions. The method is based on fluorescence of an oriented mem
brane probe excited by a polarized evanescent field created by total intern
al reflection (TIR) illumination. The fluorescent carbocyanine dye dil-C-18
-(3) (dil) has previously been shown to embed in the lipid bilayer of cell
membranes with its transition dipoles oriented nearly in the plane of the m
embrane. The membrane-embedded dil near the cell-substrate interface can be
fluorescently excited by evanescent field light polarized either perpendic
ular or parallel to the plane of the substrate coverslip. The excitation ef
ficiency from each polarization depends on the membrane orientation, and th
us the ratio of the observed fluorescence excited by these two polarization
s vividly shows regions of microscopic and submicroscopic curvature of the
membrane, and also gives information regarding the fraction of unoriented d
il in the membrane. Both a theoretical background and experimental Verifica
tion of the technique is presented for samples of 1) oriented dil in model
lipid bilayer membranes, erythrocytes, and macrophages; and 2) randomly ori
ented fluorophores in rhodamine-labeled serum albumin adsorbed to glass, in
rhodamine dextran solution, and in rhodamine dextran-loaded macrophages. S
equential digital images of the polarized TIR fluorescence ratios show spat
ially-resolved time-course maps of membrane orientations on dil-labeled mac
rophages from which low visibility membrane structures can be identified an
d quantified. To sharpen and contrast-enhance the TIR images, we deconvolut
ed them with an experimentally measured point spread function. Image deconv
olution is especially effective and fast in our application because fluores
cence in TIR emanates from a single focal plane.