We developed an approach for focused gallium-ion beam scanning electron microscopy with energy filtered detection of backscattered electrons to create near isometric voxels for high-resolution whole cell visualization. Specifically, this method allowed us to create three-dimensional volumes of high-pressure frozen, freeze-substituted Saccharomyces cerevisiae yeast cells with pixel resolutions down to 3 nm/pixel in x, y and z, supported by both empirical data and Monte Carlo simulations. As a result, we were able to segment and quantify datasets of numerous targeted subcellular structures/organelles at high-resolution, including the volume, volume percentage and surface area of the endoplasmic reticulum, cell wall, vacuoles, vacuoles, and mitochondria from an entire cell. Sites of mitochondrial and endoplasmic reticulum interconnectivity were readily identified in rendered datasets. The ability to visualize, segment and quantify entire eukaryotic cells at high-resolution (potentially sub-5 nanometers isotropic voxels) will provide new perspectives and insights of the inner workings of cells.
For more information, request a copy of the 2012 Journal of BioTechniques Article, which was selected as cover story, editor's pick, and paper of the year.