In biological research, different imaging methods unveil samples' mysteries, each with its own strengths and limitations. Light microscopy excels in live cell imaging but lacks resolution, while electron microscopy (EM) delves deeper into subcellular structures. In comparison to transmission electron microscopes (TEM), scanning electron microscopy (SEM)-based techniques opens a range of possibilities enabling scientists to overcome typical experimental limitations such as limited field-of-view and small sample volume size to better understand ultrastructural details. Expanding possibilities by combining nanometer resolution with a larger 3D context. One prominent volume EM (vEM) method is serial block-face SEM (SBF-SEM), a technology first developed and communicated by Denk et al. in 2004 [ 1]. In SBF-SEM, an ultramicrotome inside the SEM chamber cuts 15 - 30 nm thick sections from a resin-embedded sample block. The exposed sample surface is imaged with an electron beam, then new sections are cut away with a diamond knife, and the newly exposed block-face surface is imaged. This cutting and imaging process is repeated until the structure of interest is completely imaged. SBF-SEM's ultramicrotome-assisted approach captures 3D data sets for segmenting, visualizing, and analyzing cellular structures. Recent advances, like Focal Charge Compensation for challenging samples and workflow automation for unattended imaging, have further extended SBF-SEM's applications. The illustration in figure 1 outlines the SBF-SEM workflow. Robustness and automation of this technique even allow imaging and tracking of cells with extended protrusions over a long range, e.g., the investigation of neuronal network architecture as performed in Connectomics. Figure 1B and C show the reconstruction of a typical SBF-SEM data set of a mouse brain and the segmentation of such a volume stack. We would like to show the potential of a new end-to-end solution for serial block-face imaging including the latest technical achievements in hardware and software.
Figure 1:
(A) Principle of serial block-face imaging. B) 3D reconstruction of a mouse brain sample acquired with SBF-SEM C) Segmentation of a reconstructed mouse brain data set: blood vessel (red), nuclei (cyan), neurons (blue). Sample Courtesy of Christel Genoud, Université de Lausanne, EMF, 1015 Lausanne, Switzerland.
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