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SUMMARY Variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA‐TIRFM images are collected from well‐spread bovine aortic endothelial cells (BAEC) stained with a membrane‐bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64°, both the dorsal and ventral membranes were illuminated, while images excited above 66° illuminated only focal contacts. Above 74° the fluorescence of focal contacts was dominated by background noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 ± 13 nm. Focal contact areas estimated by TIRFM (1·3 ± 0·7 μm2) agreed closely with areas observed by immunofluorescence staining of vinculin (1·5 ± 0·3 μm2).

The effect of substrate surface hydrophobicity on fibronectin (Fn) adsorption and endothelial cell adhesion strength was studied. Bovine aortic endothelial cells (BAEC) were plated for 2 h with and without preadsorbed Fn on slides coated with homopolymers and copolymers of hydrophilic polyhydroxyethylmethacrylate (polyHEMA) and hydrophobic polyethylmethacrylate (polyEMA). The polarity of the substrate was determined by Wilhelmy plate contact angle. The amount of adsorbed Fn was determined using 125I-labeled Fn. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to detect gross conformational changes of adsorbed Fn on polyHEMA or polyEMA. BAEC were cultured in serum-free medium for 2 h and subjected to a brief exposure of laminar flow in a variable-height flow chamber that provided a range of shear stresses of 15-185 dynes/cm2. The critical shear stress to detach 50% of the cells increased with increasing EMA content to a maximum at 20% HEMA/80% EMA copolymer irrespective of the presence of preadsorbed Fn. However, the critical force increased even though there were similar amounts of Fn adsorbed on all substrates. ATR-FTIR spectroscopy showed only minor changes in beta-sheet structure of Fn adsorbed to polyHEMA and polyEMA. These results show that the force to detach cells did not increase solely with increasing amounts of adsorbed Fn; rather, these results indicate a more complex interplay involving both the amount and conformation of adsorbed Fn.

A simplified model of TIRF optics was used to quantitate the relative membrane/substratum separation distances from the spatial pattern of TIRF image brightness. Phase-contrast and total internal reflection fluorescence microscopy (TIRFM) images were collected of bovine aortic endothelial cells (BAEC) plated onto glass microscope slides for 15 min, 30 min and 24 h. BAEC adherent for 15 min showed an absence of a focal contact morphology, with the region of closest apposition beneath the cell center. After 30 min, multiple contacts with the surface were established and the morphology became more irregular. BAEC attached for 24 h showed well-defined focal contact regions aligned in characteristically striated patterns. The relative distance between closest and farthest membrane/substratum separations are consistent with reported distance between focal and matrix contacts. Topographical maps of membrane/substratum separation distances over the entire ventral surface of the plated cells were constructed to demonstrate the utility of quantitative TIRF microscopy.

holds B.S. and M.S. degrees in Mechanical Engineering from Clarkson University, an MBA from the New York Institute of Technology, and a Ph.D. degree in Civil and Environmental Engineering from U.C. Davis. She is currently an associate professor in the Department of Civil Engineering at the University of the Pacific. Dr. Saviz' research interests include measurement and modeling of hydrodynamics, water quality, and sediment transport in surface water systems. She has also served as co-principal investigator on several studies to develop and implement strategies to enhance the quality of engineering education. Jeff Burmeister, University of the Pacific Jeffrey S. Burmeister earned his B.S. in mechanical engineering from the University of Delaware in 1988 and his Ph.D. in biomedical engineering from Duke University in 1995. He has been a faculty member of the Department of Mechanical Engineering at the University of the Pacific since 2002 and presently is Assistant Professor and Director of the Bioengineering Program.