ENDOTHELIAL-CELL ADHESION IN REAL-TIME - MEASUREMENTS INVITRO BY TANDEM SCANNING CONFOCAL IMAGE-ANALYSIS
ENDOTHELIAL-CELL ADHESION IN REAL-TIME - MEASUREMENTS INVITRO BY TANDEM SCANNING CONFOCAL IMAGE-ANALYSIS
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DOI：
10.1172/jci116503
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发表时间：
1993-06-01
影响因子：
15.9
通讯作者：
GRIEM, ML
中科院分区：
文献类型：
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作者：
DAVIES, PF;ROBOTEWSKYJ, A;GRIEM, ML
内皮细胞中细胞-基质粘附的真实的时间测量通过近腔细胞表面的焦点接触（焦点粘附）位点的串联扫描共聚焦显微镜获得。在活细胞中清晰地定义了焦点接触位点（低辐射水平），使得图像可以被增强、数字化并与其他细胞细节分离。病灶接触部位是细胞-基质粘附的主要决定因素。（a）焦点接触面积和（B）接触的紧密度（反向辐射）的测量用于名义上定义单个细胞或细胞场的粘附，并记录真实的时间内细胞粘附的自发和诱导变化。虽然在每个焦点接触的中心或附近注意到细胞膜和基质之间的接触稍微紧密，但整个粘附区域的分离距离始终< 50 nm。连续图像的减法显示在< 1 min的时间段内未受干扰的细胞中单个焦点粘附的连续自发重塑。在延长的时间段内计算的整个细胞的细胞粘附变化<10%。当细胞骨架的稳定性受损，暴露于细胞松弛素或当细胞暴露于蛋白水解酶，内皮细胞粘附迅速下降。在单个细胞、细胞组和单个病灶粘连的水平上记录了这种变化。在这两个未受干扰和操纵的细胞，重塑和细胞粘附特性的动态变化很大，在同一个细胞内的个别网站之间，现有的网站和新的外观消失往往发生在几分钟内，而相邻的网站进行最小remodeling.Tandem扫描共聚焦显微镜图像分析活细胞在真实的时间提供重复的空间，时间和细胞粘附的定量信息。这种方法应该允许更精确的定量分析，以在活细胞中的细胞外基质，粘附蛋白，整合素和细胞骨架之间的相互作用。
Real time measurements of cell-substratum adhesion in endothelial cells were obtained by tandem scanning confocal microscopy of sites of focal contact (focal adhesions) at the abluminal cell surface. Focal contact sites were sharply defined (low radiance levels) in the living cell such that the images could be enhanced, digitized, and isolated from other cellular detail. Sites of focal contact are the principal determinant of cell-substratum adhesion. Measurements of (a) the focal contact area and (b) the closeness of contact (inverse radiance) were used to nominally define the adhesion of a single cell or field of cells, and to record spontaneous and induced changes of cell adhesion in real time.The topography of focal contacts was estimated by calculating separation distances from radiance values using a calibration technique based on interference ring optics. While slightly closer contact was noted between the cell membrane and substratum at or near the center of each focal contact, separation distances throughout the adhesion regions were always < 50 nm.Subtraction of consecutive images revealed continuous spontaneous remodeling of individual focal adhesions in unperturbed cells during periods of < 1 min. Despite extensive remodeling of focal contact sites, however, cell adhesion calculated for an entire cell over extended periods varied by < 10%. When cytoskeletal stability was impaired by exposure to cytochalasin or when cells were exposed to proteolytic enzyme, endothelial adhesion declined rapidly. Such changes were recorded at the level of single cells, groups of cells, and at single focal adhesions. In both unperturbed and manipulated cells, the dynamics of remodeling and cell adhesion characteristics varied greatly between individual sites within the same cell; disappearance of existing sites and appearance of new ones often occurred within minutes while adjacent sites underwent minimal remodelling.Tandem scanning confocal microscopy image analysis of living cells in real time provides repetitive spatial, temporal, and quantitative information about cell adhesion. Such an approach should allow more precise quantitative analyses to be made of the interactions between extracellular matrix, adhesion proteins, integrins, and the cytoskeleton in the living cell.