QUANTITATIVE-ANALYSIS OF VARIABLE-ANGLE TOTAL INTERNAL-REFLECTION FLUORESCENCE MICROSCOPY (VA-TIRFM) OF CELL SUBSTRATE CONTACTS
QUANTITATIVE-ANALYSIS OF VARIABLE-ANGLE TOTAL INTERNAL-REFLECTION FLUORESCENCE MICROSCOPY (VA-TIRFM) OF CELL SUBSTRATE CONTACTS
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DOI：
10.1111/j.1365-2818.1994.tb03426.x
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发表时间：
1994-01-01
期刊：
影响因子：
--
通讯作者：
REICHERT, WM
中科院分区：
文献类型：
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作者：
BURMEISTER, JS;TRUSKEY, GA;REICHERT, WM
可变角度全内反射荧光显微镜（VA-TIRFM）允许控制的变化的照明深度与测量膜/基板分离距离和焦点接触的大小的潜力。VA-TIRFM图像收集自用膜结合碳菁染料染色的良好铺展的牛主动脉内皮细胞（BAEC）。定量测定的绝对膜/基板分离距离和个人的焦点接触面积尝试使用一个简化模型的TIRFM光学。远角略大于64度的临界角，背侧和腹侧膜都被照亮，而高于66度激发的图像仅照亮焦点接触。在74度以上，焦点接触的荧光由背景噪声主导。不考虑背景强度而直接应用简化的光学模型是不能令人满意的。然而，在工作范围内对背景荧光进行校正和对未转换数据进行非线性回归，得到的焦点接触分离距离为24 +/- 13 nm。TIRFM估计的局部接触面积（1.3 +/- 0.7 μ m（2））与黏着斑蛋白免疫荧光染色观察到的面积（1.5 +/- 0.3 μ m（2））非常一致。
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 dis distances and individual focal contact area are attempted using a simplified model of TIRFM optics. Far 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 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 mu m(2)) agreed closely with areas observed by immunofluorescence staining of vinculin (1.5 +/- 0.3 mu m(2)).