Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells
Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells
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DOI：
10.1016/s0006-3495(00)76724-5
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发表时间：
2000-04-01
影响因子：
3.4
通讯作者：
Reichert, WM
中科院分区：
文献类型：
--
作者：
Mathur, AB;Truskey, GA;Reichert, WM
本文介绍了原子力显微镜（AFM）和全内反射荧光显微镜（TIRFM）相结合的方法，研究了力从根尖细胞膜向基底细胞膜的传递。将Bioscope AFM安装在倒置显微镜上，倒置显微镜的舞台配置为荧光标记的人脐静脉内皮细胞（HUVECs）的TIRFM成像。通过变角TIRFM实验，标定了倏逝波穿透深度对耦合角的影响。通过收集整个根尖细胞表面的一组力曲线，获得了细胞力学性能的测量。将力压痕曲线与弹性模型进行线性回归拟合，得到核上的弹性模量为7.22 +/- 0.46 kPa，核附近细胞体上的弹性模量为2.97 +/- 0.79 kPa，边缘附近细胞体上的弹性模量为1.27 +/- 0.36 kPa。应力传递是通过成像的响应基底表面局部力应用在根尖表面。当施加0.3 ~ 0.5 nN的力时，焦点触点的位置和接触面积发生变化。与施加力之前的接触面积相比，当力从细胞核中移除时，焦点接触面积显著增加（p < 0.01）。三次涂抹前后，病灶接触覆盖面积无明显变化。结果表明，细胞将局部应力从根尖全局转移到基表面，导致基表面接触的重排。
This paper describes the combined use of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) to examine the transmission of force from the apical cell membrane to the basal cell membrane. A Bioscope AFM was mounted on an inverted microscope, the stage of which was configured for TIRFM imaging of fluorescently labeled human umbilical vein endothelial cells (HUVECs). Variable-angle TIRFM experiments were conducted to calibrate the coupling angle with the depth of penetration of the evanescent wave. A measure of cellular mechanical properties was obtained by collecting a set of force curves over the entire apical cell surface. A linear regression fit of the force-indentation curves to an elastic model yields an elastic modulus of 7.22 +/- 0.46 kPa over the nucleus, 2.97 +/- 0.79 kPa over the cell body in proximity to the nucleus, and 1.27 +/- 0.36 kPa on the cell body near the edge. Stress transmission was investigated by imaging the response of the basal surface to localized force application over the apical surface. The focal contacts changed in position and contact area when forces of 0.3-0.5 nN were applied. There was a significant increase in focal contact area when the force was removed (p < 0.01) from the nucleus as compared to the contact area before force application. There was no significant change in focal contact coverage area before and after terce application over the edge. The results suggest that cells transfer localized stress from the apical to the basal surface globally, resulting in rearrangement of contacts on the basal surface.