Cell membrane orientation visualized by polarized total internal reflection fluorescence

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.