Manganese (Mn) accumulates at a higher level in the pancreas than in any other organs when excess Mn is administered to the rat. The present study is carded out to analyze the intracellular localization of Mn existed in the pancreatic cell of Mn‐treated rats. Transmission electron microscope and X‐ray micro‐analysis connected with a rapid freezing fixation technique showed that a large amount of Mn was localized in tysosomal particles of the pancreatic cell of Mn‐treated rats. The Mn‐rich particles disappeared when the element‐administration was discontinued, showing that the accumulation of Mn is reversible. To confirm that Mn is in the lysosomes, a centrifugal subcellular‐fractionation and a neutron activation analysis were carried out. The result indicated that much Mn existed in the lysosomal fraction.
Array tomography (AT) provides three-dimensional (3D) information by observing serial sections using scanning electron microscopy (SEM). Compared with serial block-face SEM and focused ion beam (FIB)/SEM, AT has several advantages: (a) High lateral resolution, (b) Standard staining with uranium and lead, allowing easy comparison of images captured by SEM and transmission electron microscopy (TEM), (c) Repetitive observations of samples, enabling hierarchical analysis from low magnification to high magnification, and (d) Low installation cost. In many cases, however, images of serial sections have been taken manually with labor and time. In addition, manual segmentation has been usually required. To solve these issues, we developed automatic image capturing software for AT with SEM. We also tried to use deep neural network to assist segmentation of the images.In Figure 1, flow diagram of observation and analysis is shown. Mouse cerebellum cortex was fixed with glutaraldehyde and osmium tetra oxide, and was embedded in epoxy resin. Then serial sections were cut using ultra microtome and scooped on silicon substrate, and they were stained with uranyl acetate and lead citrate. SEM (JSM-7800F and JSM-7900F; JEOL Ltd.) was used for capturing images.A software was developed for automatic capturing of SEM images in each serial section at the same position following manual identification of the corresponding apex of serial sections. Convolutional neural network algorithm was used to extract cell nuclei in each image. Since such an automatic segmentation was not perfectly completed, we manually corrected the data after the automatic segmentation. Then, 3D image was reconstructed by the stack of images from serial sections, and evaluated with quantitative analysis.In Figure 2, images of cell nuclei in various cells are shown. Since the contrast of images was similar to that obtained by TEM, microstructures including cell nuclei were easily identified. A 3D-reconstructed image is shown in Figure 3. The automatic image capturing software and deep neural network analysis brought about easier procedure for the segmentation and quantitative analysis. Such a system for analysis of SEM images with AT will realize to be used for pathological diagnosis as well as basic biology.References:[1] Micheva et. al, Neuron, 55 (2007), p. 25.
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