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

发展了一种细胞旋转系统，可以从不同角度获得的细胞图像精确地观察细胞的三维微结构。在该系统中，通过在带有浸水物镜的直立显微镜下轻轻地吸入细胞表面，将标本细胞与微吸管保持在一起，并绕着设置成垂直于显微镜光轴的微吸管的轴线旋转。将吸管安装在3D电动微操作器上，并根据安装在显微镜上的CCD摄像机拍摄的图像控制吸管尖端的位置。在吸头上涂上黑色墨水，通过对吸头图像的二值化来确定吸头在聚焦(X-Y)平面上的位置。尖端位置信号被馈送到在X和Y方向上控制微操作器的比较器。通过改变吸管的Z向位置，使吸头的二值化面积保持最小，从而使吸头保持聚焦。用SYTO13和DiI分别标记培养的牛胸主动脉平滑肌细胞的细胞核和细胞膜，并连接到微管顶端。从0°到18°，每隔9°通过细胞的光学切片构建全聚焦图像。从这些泛聚焦图像中获得了细胞核和细胞膜的三维图像。这些重建图像表明，Wirth胰酶底物上的细胞分离器的细胞核呈圆形，而福尔马林固定在底物上的细胞的细胞核被拉长和扁平。本发明用于利用细胞旋转系统重建细胞微结构的方法将有助于对细胞形态的定量3D分析。

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.