Development of automatic electron microscope for tomography and application to cell biology

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We have developed an automatic electron microscope for tomogaraphy on the basis of a Hitachi H-9500 EM whose acceleration voltage is 200 or 300 kV. This EM has following features ; (a)the pole piece designed for high angle tilt up to 70deg, (b)the eucentric goniometer with five adjustable axes, (c)the high sensitivity CCD camera with 2k x 2k pixels (TemCam-F224S, TVIPS) and (d)the automatic image acquisition for a tilt-series. The main points of the development are the interface between the EM and the camera, and the software EM-menu of TVIPS enables us to operate the auto-tracking and the auto-focusing for the EM image.After acquisition of the tilt-series EM images, the tomogram is reconstructed with the software IMOD developed at Colorado University. Then, we added a function that the arraignment of tilt-series images carries out automatically without fiducial markers such as artificial gold particles. That is, we developed new software named "CA markers" that can choose the feature points and truck the each point among the tilt-series images.With the system and software, we reconstructed 3D images of a few samples such as microvillus of duodenum, HIV invaded into cell membrane and nuclear of malignant cells.