大気圧走査電子顕微鏡(ASEM)による水中免疫電顕法 : タンパク質複合体のダイナミックな離合集散と細胞内移動  [in Japanese] Immuno Correlative Microscopy in Solution Using Atmospheric Scanning Electron Microscope (ASEM) : Observation of Dynamic Rearrangements of Molecular Complexes  [in Japanese]

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Abstract

<p>タンパク質の細胞内分布は高度に制御され,刺激に応じてダイナミックに変化する.免疫電顕法はタンパク質の精妙な細胞内配置による機能の解明に貢献してきた.膜タンパク質が,刺激後に細胞内膜から細胞膜へ数秒以内に移動する例などが近年多く見つかってきており,多要素の変化を時間経過とともに解析する必要が増してきている.すなわち,従来よりも迅速な試料作製と観察が可能な,いわゆる高スループットな電子顕微鏡が求められている.大気圧走査電子顕微鏡(ASEM)は,培養ディッシュ内の水溶液中試料をディッシュ底の電子線透過薄膜越しに倒立走査電顕で観察する新しいタイプの電顕である.試料は脱水処理なしに,蛍光抗体法と同様に手早く作製できる.しかも細胞や組織の抗原分布は薄膜から2~3 μmの厚さで観察可能である.さらに上方の光顕による蛍光像との対比によって,タンパク質複合体の形成など多分子相関の詳細な観察を実現している.</p>

<p>High-resolution observation of cells and tissues is principally carried out by electron microscopy (EM), although standard EM requires the sample be in vacuum. In the ASEM, an inverted SEM observes the wet sample from beneath an open dish while an optical microscope (OM) observes it from above. The disposable dish with a silicon nitride (SiN) film window can hold a few milliliters of culture medium, and allows various types of cells to be cultured in a stable environment. The use of this system for <i>in situ</i> correlative OM/SEM immuno-microscopy is explored, the efficiency of the required dual-tagged labeling assessed and the imaging capabilities of the ASEM documented. We have visualized a dynamic string-like gathering of STIM1 on the ER in Jurkat T cells in response to Ca<sup>2+</sup> store depletion. We have also visualized filamentous-actin (F-actin) and tubulin in the growth cones of primary-culture neurons as well as in synapses. Further, radially running actin fibers were shown to partly colocalize with concentric bands of the Ca<sup>2+</sup> signaling component Homer1c in the lamellipodia of neuron primary culture growth cones. After synapse formation, neurite configurations were drastically rearranged; a button structure with a fine F-actin frame faces a spine with a different F-actin framework.</p>

Journal

  • KENBIKYO

    KENBIKYO 48(2), 107-112, 2013-08-30

    The Japanese Society of Microscopy

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