title: Rapid specimen preparation to improve the throughput of electron microscopic volume imaging for three-dimensional analyses of subcellular ultrastructures with serial block-face scanning electron microscopy.
abstract: Serial block-face imaging using scanning electron microscopy enables rapid observations of three-dimensional ultrastructures in a large volume of biological specimens. However, such imaging usually requires days for sample preparation to reduce charging and increase image contrast. In this study, we report a rapid procedure to acquire serial electron microscopic images within 1 day for three-dimensional analyses of subcellular ultrastructures. This procedure is based on serial block-face with two major modifications, including a new sample treatment device and direct polymerization on the rivets, to reduce the time and workload needed. The modified procedure without uranyl acetate can produce tens of embedded samples observable under serial block-face scanning electron microscopy within 1 day. The serial images obtained are similar to the block-face images acquired by common procedures, and are applicable to three-dimensional reconstructions at a subcellular resolution. Using this approach, regional immune deposits and the double contour or heterogeneous thinning of basement membranes were observed in the glomerular capillary loops of an autoimmune nephropathy model. These modifications provide options to improve the throughput of three-dimensional electron microscopic examinations, and will ultimately be beneficial for the wider application of volume imaging in life science and clinical medicine.
authors: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo-City, Yamanashi, 409-3898, Japan.
authors: Truc Quynh
authors: TQ
authors: Thai
authors: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo-City, Yamanashi, 409-3898, Japan.
authors: Huy Bang
authors: HB
authors: Nguyen
authors: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo-City, Yamanashi, 409-3898, Japan.
authors: Sei
authors: S
authors: Saitoh
authors: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo-City, Yamanashi, 409-3898, Japan.
authors: Bao
authors: B
authors: Wu
authors: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo-City, Yamanashi, 409-3898, Japan.
authors: Yurika
authors: Y
authors: Saitoh
authors: Department of Occupational Therapy, Health Science University, 7187 Kodachi, Fujikawaguchiko, Yamanashi, 401-0380, Japan.
authors: Satoshi
authors: S
authors: Shimo
authors: Department of Histology and Cytology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, 44519, Egypt.|Laboratory of Anatomy, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo City, Hokkaido, 060-0818, Japan.
authors: Yaser Hosny Ali
authors: YH
authors: Elewa
authors: Laboratory of Anatomy, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo City, Hokkaido, 060-0818, Japan.
authors: Osamu
authors: O
authors: Ichii
authors: Laboratory of Anatomy, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo City, Hokkaido, 060-0818, Japan.
authors: Yasuhiro
authors: Y
authors: Kon
authors: Department of Pathology, Tohoku University, Sendai, Miyagi, 980-8575, Japan.
authors: Takashi
authors: T
authors: Takaki
authors: Department of Pathology, Tohoku University, Sendai, Miyagi, 980-8575, Japan.
authors: Kensuke
authors: K
authors: Joh
authors: Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo-City, Yamanashi, 409-3898, Japan. oonon-tky@umin.ac.jp.|Center for Multidisciplinary Brain Research, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8787, Japan. oonon-tky@umin.ac.jp.
authors: Nobuhiko
authors: N
authors: Ohno
mesh_terms: D000818:Animals; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007668:Kidney / Q000473:pathology; D008810:Mice, Inbred C57BL; D019463:Mice, Inbred MRL lpr; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*; D013347:Subcellular Fractions / Q000648:ultrastructure
keywords: High throughput; Serial block-face scanning electron microscopy; Tissue preparation; Uranyl acetate