title: Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography.
abstract: Electron tomography using a high-voltage electron microscope (HVEM) provides three-dimensional information about cellular components in sections thicker than 1 μm, although in bright-field mode image degradation caused by multiple inelastic scattering of transmitted electrons limit the attainable resolution. Scanning transmission electron microscopy (STEM) is believed to give enhanced contrast and resolution compared to conventional transmission electron microscopy (CTEM). Samples up to 1 μm in thickness have been analyzed with an intermediate-voltage electron microscope because inelastic scattering is not a critical limitation, and probe broadening can be minimized. Here, we employed STEM at 1 MeV high-voltage to extend the useful specimen thickness for electron tomography, which we demonstrate by a seamless tomographic reconstruction of a whole, budding Saccharomyces cerevisiae yeast cell, which is ~3 μm in thickness. High-voltage STEM tomography, especially in the bright-field mode, demonstrated sufficiently enhanced contrast and intensity, compared to CTEM tomography, to permit segmentation of major organelles in the whole cell. STEM imaging also reduced specimen shrinkage during tilt-series acquisition. The fidelity of structural preservation was limited by cytoplasmic extraction, and the spatial resolution was limited by the relatively large convergence angle of the scanning probe. However, the new technique has potential to solve longstanding problems of image blurring in biological specimens beyond 1 μm in thickness, and may facilitate new research in cellular structural biology.
authors.affiliation: National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan. Electronic address: kazum@nips.ac.jp.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Murata
authors.affiliation: Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Esaki
authors.affiliation: Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan.
authors.forename: Teru
authors.initials: T
authors.lastname: Ogura
authors.affiliation: Ecotopia Science Institute, Nagoya University, Nagoya, Aichi 464-8603, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Arai
authors.affiliation: Ecotopia Science Institute, Nagoya University, Nagoya, Aichi 464-8603, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Yamamoto
authors.affiliation: Ecotopia Science Institute, Nagoya University, Nagoya, Aichi 464-8603, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Tanaka
mesh_terms: D055032:Electron Microscope Tomography / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure*
keywords: Electron tomography; High-voltage electron microscopy; Organelle; Scanning transmission electron microscopy; Thick specimen; Yeast