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

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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.