title: Immuno-electron microscopy of primary cell cultures from genetically modified animals in liquid by atmospheric scanning electron microscopy.
abstract: High-throughput immuno-electron microscopy is required to capture the protein-protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell's plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.
authors.affiliation: 1 Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Kinoshita
authors.affiliation: 2 Department of Virology III, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan.
authors.forename: Yosio
authors.initials: Y
authors.lastname: Mori
authors.affiliation: 1 Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Hirano
authors.affiliation: 3 Department of Bacteriology, The Jikei University School of Medicine, Tokyo 105-8461, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Sugimoto
authors.affiliation: 3 Department of Bacteriology, The Jikei University School of Medicine, Tokyo 105-8461, Japan.
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Okuda
authors.affiliation: 4 Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Matsumoto
authors.affiliation: 5 Suntory Global Innovation Center, Research Institute, 5-2-5 Yamazaki, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Namiki
authors.affiliation: 6 Biomedical Research Institute and Information Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Ebihara
authors.affiliation: 6 Biomedical Research Institute and Information Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kawata
authors.affiliation: 7 JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Nishiyama
authors.affiliation: 6 Biomedical Research Institute and Information Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Sato
authors.affiliation: 7 JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Suga
authors.affiliation: 5 Suntory Global Innovation Center, Research Institute, 5-2-5 Yamazaki, Shimamoto-cho, Mishima-gun, Osaka 618-0001, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Higashiyama
authors.affiliation: 8 Laboratory of Microbial Technology, Department of Bioscience and Biotechnology, Division of Applied Molecular Microbiology and Biomass Chemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Sonomoto
authors.affiliation: 3 Department of Bacteriology, The Jikei University School of Medicine, Tokyo 105-8461, Japan.
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Mizunoe
authors.affiliation: 1 Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Nishihara
authors.affiliation: 6 Biomedical Research Institute and Information Technology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Chikara
authors.initials: C
authors.lastname: Sato
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D003584:Cytological Techniques / Q000379:methods*; D004330:Drosophila / Q000166:cytology*; D004707:Endoderm / Q000166:cytology; D056546:Lactobacillales / Q000166:cytology / Q000502:physiology; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D016253:Microscopy, Immunoelectron / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000502:physiology; D061251:Primary Cell Culture; D012412:Rubella virus / Q000502:physiology; D013194:Staining and Labeling / Q000379:methods; D014779:Virus Replication