DepartmentMolecular Cell Biology
Publication date1-Jul-2014

Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography.
Kazuyoshi Murata, Masatoshi Esaki, Teru Ogura, Shigeo Arai, Yuta Yamamoto, and Nobuo Tanaka

Ultramicroscopy 146, 39-45, 2014

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 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 1μm in thickness, and may facilitate new research in cellular structural biology.
This study was supported in part by the program of the Joint Usage/Research Center for Developmental Medicine, IMEG, Kumamoto University.



Figure. Segmented surface-rendered model of wild type yeast cells from scanning transmission electron microscopy tomography.
 Plasma membranes (green), nucleus (red), vacuoles (blue)], and mitochondria (yellow) were clearly observed.