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

Electron tomography using a high voltage electron microscope (HVEM) provides three-dimensional information about cellular components in sections thicker than 1 pm, although in bright held 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 mu m in thickness have been analyzed with an intermediate voltage electron microscope because inelastic scattering is nor 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 Lomographic reconstruction of a whole, budding Saccharomyces cerevisiae yeast cell, which is similar to 3 mu m 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 pill in thickness, and may facilitate new research in cellular structural biology. (C) 2014 Elsevier B.V. All rights reserved.