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-C(18)-(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.
Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about relevant chemical kinetic rates in vivo. Total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP), an established technique previously demonstrated to measure reversible biomolecular kinetic rates at surfaces in vitro, is extended here to measure reversible biomolecular kinetic rates of actin at the cytofacial (subplasma membrane) surface of living cells. For the first time, spatial imaging (with a charge-coupled device camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging produces both spatial maps of kinetic parameters (off-rates and mobile fractions) and estimates of kinetic correlation distances, cell-wide kinetic gradients, and dependences of kinetic parameters on initial fluorescence intensity. For microinjected rhodamine actin in living cultured smooth muscle (BC3H1) cells, the unbinding rate at or near the cytofacial surface of the plasma membrane (averaged over the entire cell) is measured at 0.032 +/- 0.007 s(-1). The corresponding rate for actin marked by microinjected rhodamine phalloidin is very similar, 0.033 +/- 0.013 s(-1), suggesting that TIR/FRAP is reporting the dynamics of entire filaments or protofilaments. For submembrane fluorescence-marked actin, the intensity, off-rate, and mobile fraction show a positive correlation over a characteristic distance of 1-3 microm and a negative correlation over larger distances greater than approximately 7-14 microm. Furthermore, the kinetic parameters display a statistically significant cell-wide gradient, with the cell having a "fast" and "slow" end with respect to actin kinetics.
energies of about 20 GeV. Fortunately, recent experiments [5][6][7][8] no polarization was lost. This supports the conjecture that a Siberian snake can be varying the snake either once, twice or ten times; we found with good precision that between about 0 and 25% at 370 MeV. We measured the beam polarization after with a superconducting solenoid; this combination allowed varying the snake strength strengths. The snake consisted of two rampable warm solenoid magnets in series a partial Siberian snake at 370 MeV, where the spin tune, 1/,, is 2% for all snake A recent experiment in the IUCF cooler ring studied the adiabatic turn-on of
Binding/unbinding kinetic rates of cytoskeletal proteins at the cytofacial surface of the plasma membrane of smooth muscle cells in culture are measured and imaged by total internal reflectionlfluorescence recovery after photobleaching (TIRIFRAP) microscopy. Cells are first injected with either rhodamine monomeric actin or rhodamine phalloidin, which binds to polymeric actin. A TIR beam, which illuminates approximately 80 rim deep into the cell, is then used to preferentially observe the labeled actin in the vicinity of the membrane. Fluorophores at cell-substrate contact regions within the evanescent field illumination are then photobleached by a prolonged flash. The subsequent recovery, excited by a series of briefer flashes at I or 6 frames per minute, is recorded by a cooled CCD. The resulting stack of images can be curve-fit pixel-by-pixel to produce a spatially resolved image of the unbinding rate (the reciprocal of residency time) of protein reversibly adsorbed at the submembrane surface. For rhodamine actin and for rhodamine phalloidin, the two ways of marking actin in the cell, the average characteristic unbinding times are somewhat different: 308 142 sec and 833 140 sec, respectively. The spatially resolved images of rhodamine phalloidin, pseudo-colorized to show average unbinding rates at each pixel, reveal a considerable variation over the cell, ranging over an order of magnitude.
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