Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography

• Published in: Ultramicroscopy Vol. 146; pp. 39 - 45
• Main Authors: Murata, Kazuyoshi, Esaki, Masatoshi, Ogura, Teru, Arai, Shigeo, Yamamoto, Yuta, Tanaka, Nobuo
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2014
• Subjects: Cellular; Electron Microscope Tomography - methods; Electron microscopes; Electron tomography; High-voltage electron microscopy; Image Processing, Computer-Assisted; Imaging; Inelastic scattering; Microscopy, Electron, Scanning Transmission - methods; Organelle; Saccharomyces cerevisiae; Saccharomyces cerevisiae - ultrastructure; Scanning transmission electron microscopy; Thick specimen; Tomography; Yeast; ADF; Yeast; BF; Thick specimen; MPL; Electron tomography; STEM; ET; HVEM; Organelle; High-voltage electron microscopy; Scanning transmission electron microscopy; TEM; HAADF
• ISSN: 0304-3991; 1879-2723
• DOI: 10.1016/j.ultramic.2014.05.008

Electron tomography using a high-voltage electron microscope (HVEM) provides three-dimensional information about cellular components in sections thicker than 1μm, 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 1μm 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 1MeV 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 ~3μm 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 1μm in thickness, and may facilitate new research in cellular structural biology. •High voltage TEM and STEM tomography were compared to visualize whole yeast cells.•1-MeV STEM-BF tomography had significant improvements in image contrast and SNR.•1-MeV STEM tomography showed less specimen shrinkage than the TEM tomography.•KMnO4 post-treatment permitted segmenting the major cellular components.