Studies on cell movement and cvtoskeleton by atomic force microscopy

原子力显微镜（AFM）没有透镜，只是在样品表面扫描一个尖锐的探针，提供从微米尺度到原子尺度的高分辨率样品的地形图像。在本研究中，我们将AFM应用于活体培养动物细胞的三维观察。我们使用牢牢附着在衬底上的扁平细胞，因为在扫描过程中球形细胞很容易从衬底表面分离。为了尽量减少力对细胞的损伤，成像力被调整到最弱的值。因此，活细胞的接触模式AFM提供了细胞边缘细胞突起（尖刺状突起、板足等）形状的精确信息。在细胞的上表面也可以清楚地观察到细胞膜下的细胞骨架元素的轮廓。然后，我们成功地获得活细胞的连续AFM图像，时间间隔为2-4分钟，使用流体室系统超过一个小时。一系列这些AFM图像对于检查与亚细胞细胞骨架元素相关的细胞过程的运动是有用的。由连续AFM图像制作的延时电影也证实了细胞动力学的真实性。最后，我们将AFM图像与荧光显微镜或扫描电子显微镜拍摄的图像进行了比较。数据表明，在领先的过程中，在颗粒高度存在肌动蛋白积累。

The atomic force microscope (AFM) has no lens but simply scans a sharp probing tip over a sample surface, providing topographic images of samples at high resolutions from the micrometer scale to the atomic scale. In this study, we applied AFM to the three-dimensional observation of cultured animal cells in a living state.We used flat cells attached firmly to the substrate, becauce spherical cells were easily detached from the substrate surface during scanning. To minimize force-induced damages to the cell, imaging force was adjusted to the weakest value. Thus, contact mode AFM of the living cells provided precise information on the shape of cellular processes (spike-like processes, lamellipodia, etc.) at the cellular margin. The contour of cytoskeletal elements just beneath the cell membrane was also clearly observable on the upper surface of the cells.We then succeeded in obtaining continuously AFM images of living cells for over one hour at time intervals of 2-4 min by using a fluid chamber system. A series of these AFM images were useful for examining the movements of cellular processes in relation to subcellular cytoskeletal elements. Time-lapse movies produced by sequential AFM images also verified the reality of the cellular dynamics.Wefinally compared the AFM images with images taken by fluorescent microscopy, or scanning electron microscopy. The data indicated the presence of actin accumulation in granular elevations on the leading processes.