Monitoring cell movements and volume changes with pulse-mode scanning ion conductance microscopy
Monitoring cell movements and volume changes with pulse-mode scanning ion conductance microscopy
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DOI：
10.1046/j.1365-2818.2003.01248.x
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发表时间：
2003-11-01
期刊：
影响因子：
--
通讯作者：
Dietzel, ID
中科院分区：
文献类型：
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作者：
Happel, P;Hoffmann, G;Dietzel, ID
在这里，我们描述了使用脉冲模式扫描离子电导显微镜（SICM）观察体积变化和细胞膜运动过程中的运动培养细胞在几分钟到几个小时的范围内。该显微镜是基于脉冲模式SICM先前开发的培养中的单细胞的稳定成像。我们的仪器使用电流脉冲来控制细胞表面和电极尖端之间的距离，以及后退模式，以防止在探针的横向移动过程中尖端和膜的接触。我们使用反馈控制的压电元件来定位电极，对细胞表面进行重复扫描。使用膜片钳型电极尖端，可以重复地测量细胞的高度，标准偏差为50 nm。为了量化和分离连续扫描之间发生的细胞位置和体积的变化，编写了一个程序来减去图像并计算体积变化。重复扫描的实例表明，可以使用差分图像以500 nm的横向分辨率定量监测30分钟至几小时范围内的膜运动，并且可以使用线扫描模式在限定的细胞切片处记录分钟范围内的更快运动。差异图像表明，体积变化可以影响细胞表面不均匀，突出的作用，细胞骨架的稳定细胞形状。
Here we describe the use of pulse-mode scanning ion conductance microscopy (SICM) to observe volume changes and cell membrane movements during the locomotion of cultured cells in the range of minutes to several hours. The microscope is based on the pulse-mode SICM previously developed for stable imaging of single cells in culture. Our instrument uses current pulses to control the distance between cell surface and electrode tip as well as a back-step mode to prevent contact of tip and membrane during lateral movements of the probe. We performed repeated scans of cell surfaces using feedback-controlled piezoactors to position the electrode. Using patch-clamp-type electrode tips the height of cells could reproducibly be measured with a standard deviation of 50 nm. To quantify and separate changes in cell position and volume occurring between consecutive scans, a program was written to subtract images and calculate volume changes. Examples of repeated scans show that membrane movements in the range of 30 min to a few hours can be quantitatively monitored with a lateral resolution of 500 nm using difference images and that faster movements in the range of minutes can be recorded at defined cell sections using the line scan mode. Difference images indicate that volume changes can affect cell surfaces inhomogeneously, emphazising the role of the cytoskeleton in the stabilization of cell shape.