Burmeister J S, Truskey G A, Reichert W M
Department of Biomedical Engineering, Duke University, Durham, NC 27708.
J Microsc. 1994 Jan;173(Pt 1):39-51. doi: 10.1111/j.1365-2818.1994.tb03426.x.
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 degrees, both the dorsal and ventral membranes were illuminated, while images excited above 66 degrees illuminated only focal contacts. Above 74 degrees the fluorescence of focal contacts was dominated by back-ground 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 micron2) agreed closely with areas observed by immunofluorescence staining of vinculin (1.5 +/- 0.3 microns2).
可变角度全内反射荧光显微镜（VA-TIRFM）能够控制照明深度的变化，具备测量膜/底物分离距离和粘着斑大小的潜力。VA-TIRFM图像是从用膜结合花菁染料染色的充分铺展的牛主动脉内皮细胞（BAEC）中采集的。尝试使用TIRFM光学的简化模型对绝对膜/底物分离距离和单个粘着斑面积进行定量测定。对于略大于64度临界角的角度，背侧和腹侧膜均被照亮，而在66度以上激发的图像仅照亮粘着斑。在74度以上，粘着斑的荧光以背景噪声为主。在不考虑背景强度的情况下直接应用简化光学模型并不理想。然而，对背景荧光进行校正并在工作范围内对未转换数据进行非线性回归，得出粘着斑分离距离为24±13nm。通过TIRFM估计的粘着斑面积（1.3±0.7平方微米）与通过纽蛋白免疫荧光染色观察到的面积（1.5±0.3平方微米）非常吻合。
