Immuno-electron microscopy of primary cell cultures from genetically modified animals in liquid by atmospheric scanning electron microscopy

Takaaki Kinoshita, Yosio Mori, Kazumi Hirano, Shinya Sugimoto, Ken I. Okuda, Shunsuke Matsumoto, Takeshi Namiki, Tatsuhiko Ebihara, Masaaki Kawata, Hidetoshi Nishiyama, Mari Sato, Mitsuo Suga, Kenichi Higashiyama, Kenji Sonomoto, Yoshimitsu Mizunoe, Shoko Nishihara, Chikara Sato

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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.

Original languageEnglish
Pages (from-to)469-483
Number of pages15
JournalMicroscopy and Microanalysis
Volume20
Issue number2
DOIs
Publication statusPublished - Apr 2014

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All Science Journal Classification (ASJC) codes

  • Instrumentation

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