Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM.
Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future.
Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan.
Kazumi
K
Hirano
Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan.
Takaaki
T
Kinoshita
Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Department of Molecular and Cellular Physiology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
Takeshi
T
Uemura
Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan.
Hozumi
H
Motohashi
Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan.
Yohei
Y
Watanabe
Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan.
Tatsuhiko
T
Ebihara
JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558, Japan.
Hidetoshi
H
Nishiyama
Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan.
Mari
M
Sato
JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558, Japan.
Mitsuo
M
Suga
Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan.
Yuusuke
Y
Maruyama
Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan.
Noriko M
NM
Tsuji
Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan.
Masayuki
M
Yamamoto
Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan. Electronic address: shoko@soka.ac.jp.
Shoko
S
Nishihara
Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan. Electronic address: ti-sato@aist.go.jp.
Chikara
C
Sato
D000199:Actins / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004330:Drosophila / Q000166:cytology; D006046:Gold / Q000378:metabolism; D051379:Mice; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D009474:Neurons / Q000166:cytology*; D061848:Optical Imaging / Q000379:methods*; D010587:Phagocytosis / Q000502:physiology; D061251:Primary Cell Culture / Q000379:methods*; D017655:Silicon Compounds / Q000737:chemistry; D012996:Solutions / Q000737:chemistry*
D000199:Actins; D017655:Silicon Compounds; D012996:Solutions; D006046:Gold; C032734:silicon nitride
ASEM; ClairScope; Environmental cell; Immuno-EM; Neuron