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Development of a cell rotation sytem for the observation of 3D microstructure of cells

开发用于观察细胞3D微观结构的细胞旋转系统

基本信息

批准号：
12558101
负责人：
MATSUMOTO Takeo
金额：
$ 8.45万
依托单位：
Nagoya Institute of Technology (2002)Tohoku University (2000-2001)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12558101/
关键词：
Biomechanics Cellular mechanics Cytoskeleton Organellae Microscop Cell nucleus 細胞小器管

项目摘要

A cell rotation system has been developed to observe the 3D miciostructure of cells precisely from their images obtained from various view points. In this system, a specimen cell is held with a micropipette by aspirating the cellular surface gently under an upright microscope with water immersion objectives, and is rotated around the axis of the micropipette which is set perpendicular to the optical axis of the microscope. The pipette was mounted on a 3D electric micromanipulator and the position of the pipette tip was controlled based on its image taken with a CCD video camera mounted on the microscope. The pipette tip was coated with black ink and the tip position in the focusing (X-Y) plane was determined by binarization of the tip image. The tip position signal was fed to a comparator controlling the micromanipulators in X and Y direction. The pipette tip was kept focused with a PID controller maintaining the binarized area of the pipette tip minimum by changing the Z position of the pipette. Cultured bovine thoracic aortic smooth muscle cells were stained with SYTO13 for their nucleus and DiI for their cellular membrane and attached to the micropipette tip. Pan-focus images were constructed from optical sections across a cell at every 9° from 0° to 18° . The three-dimensional images of the cell nucleus and the cell membrane were obtained from these pan-focus images. These reconstructed images indicated that the nucleus of the cell detacher from the substrate wirth trypsin was round in shape, while that of the cell fixed with formalin on the substrate was elongated and flattened. The present method for the reconstruction of the cell microstructure with the cell rotation system would be useful for quantitative 3D analysis of the cell morphology.

开发了一种细胞旋转系统，可以从不同视点获得的细胞图像中精确观察细胞的三维显微结构。在这个系统中，在一个带有水浸物镜的立式显微镜下，用微移管轻轻吸吸细胞表面，并围绕垂直于显微镜光轴的微移管轴旋转细胞标本。将移液器安装在三维电子显微操作器上，通过安装在显微镜上的CCD摄像机拍摄的移液器图像来控制移液器尖端的位置。在移液器尖端涂上黑色墨水，通过对尖端图像二值化确定其在聚焦（X-Y）平面上的位置。将尖端位置信号馈送到控制微机械臂X和Y方向的比较器。通过改变移液器的Z位置，PID控制器保持移液器尖端的二值化面积最小，从而使移液器尖端保持聚焦。用SYTO13染色牛胸主动脉平滑肌细胞的细胞核，用DiI染色牛胸主动脉平滑肌细胞的细胞膜，并贴于微移液管尖端。从0°到18°每隔9°的光学切片构建泛聚焦图像。这些泛聚焦图像获得了细胞核和细胞膜的三维图像。这些重建图像表明，与胰蛋白酶分离的细胞细胞核呈圆形，而与福尔马林固定在底物上的细胞细胞核呈细长和扁平状。利用细胞旋转系统重建细胞微观结构的方法可用于细胞形态的定量三维分析。