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

本文介绍了一种细胞旋转系统，它能从不同的角度观察细胞的三维显微结构。在该系统中，通过在具有水浸物镜的直立显微镜下轻轻地抽吸细胞表面，用微量移液管保持试样细胞，并围绕微量移液管的轴旋转，微量移液管的轴设置为垂直于显微镜的光轴。将移液器安装在3D电动显微操作器上，并基于用安装在显微镜上的CCD摄像机拍摄的移液器尖端的图像来控制移液器尖端的位置。用黑色墨水涂覆移液器吸头，并通过吸头图像的二值化确定吸头在聚焦（X-Y）平面中的位置。尖端位置信号被馈送到在X和Y方向上控制显微操作器的比较器。用PID控制器保持移液器吸头聚焦，通过改变移液器的Z位置来维持移液器吸头的二值化区域最小。培养的牛胸主动脉平滑肌细胞的细胞核用SYTO 13染色，细胞膜用DiI染色，并附着在微量移液器吸头上。从0°至18°，每9°从细胞的光学切片构建泛焦图像。从这些全焦点图像获得细胞核和细胞膜的三维图像。这些重建图像表明，从底物胰蛋白酶固定的细胞核是圆形的，而用福尔马林固定在底物上的细胞核是细长和扁平的。本发明的方法与细胞旋转系统的细胞微观结构的重建将是有用的细胞形态的定量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.