QUANTITATIVE-ANALYSIS OF VARIABLE-ANGLE TOTAL INTERNAL-REFLECTION FLUORESCENCE MICROSCOPY (VA-TIRFM) OF CELL SUBSTRATE CONTACTS

Variable-angle total internal reflection fluorescence microscopy (VA-T IRFM) allows controlled variation of the illumination depth with the p otential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA-TIRFM images are collected from well-spre ad bovine aortic endothelial cells (BAEC) stained with a membrane-boun d carbocyanine dye. Quantitative determination of absolute membrane/su bstrate separation dis distances and individual focal contact area are attempted using a simplified model of TIRFM optics. Far angles slight ly greater than the critical angle of 64 degrees, both the dorsal and ventral membranes were illuminated, while images excited above 66 degr ees illuminated only focal contacts. Above 74 degrees the fluorescence of focal contacts was dominated by background noise. Direct applicati on of the simplified optical model without accounting for background i ntensity was unsatisfactory. However, correction for background fluore scence and nonlinear regression of the untransformed data over the wor king range yielded focal contact separation distances of 24 +/- 13 nm. Focal contact areas estimated by TIRFM (1.3 +/- 0.7 mu m(2)) agreed c losely with areas observed by immunofluorescence staining of vinculin (1.5 +/- 0.3 mu m(2)).