Truskey G A, Burmeister J S, Grapa E, Reichert W M
Department of Biomedical Engineering, Duke University, Durham, NC 27706.
J Cell Sci. 1992 Oct;103 ( Pt 2):491-9. doi: 10.1242/jcs.103.2.491.
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.
使用一种简化的全内反射荧光（TIRF）光学模型，根据TIRF图像亮度的空间模式来定量测定相对膜/基质分离距离。采集了接种在玻璃显微镜载玻片上15分钟、30分钟和24小时的牛主动脉内皮细胞（BAEC）的相差显微镜图像和全内反射荧光显微镜（TIRFM）图像。贴壁15分钟的BAEC未显示出粘着斑形态，细胞中心下方为最紧密贴合区域。30分钟后，与表面建立了多个接触点，形态变得更不规则。贴壁24小时的BAEC显示出排列成特征性条纹模式的明确粘着斑区域。最接近和最远距离的膜/基质分离之间的相对距离与报道的粘着斑和基质接触之间的距离一致。构建了接种细胞整个腹面的膜/基质分离距离地形图，以证明定量TIRF显微镜的实用性。
