The nervous system is densely packed with ultrastructures that can be as thin as 40 to 50 nm in diameter (Meinertzhagen and O’Neil, 1991), and tracing these ultrastructures is required to reconstruct their local connectivity or to estimate their morphology. Light microscopic tracing is only possible via labeling a subset of neurons, and more importantly, the resolution available in light microscopy is insufficient to resolve thin ultrastructures when their diameters are substantially below the light wavelength. The application of standard heavy metals-based EM stains, on the other hand, results in relatively unbiased staining of all membranes and synapses (Peters, Palay and Webster, 1991). EM techniques also provide the spatial resolution to resolve densely packed neighboring neuronal processes for reconstructing neural circuits or estimating neuronal geometry (Denk and
There are two main types of EM techniques, transmission EM (TEM) and SEM.
TEM emits high-voltage electrons to a thin specimen, and only a fraction of electrons pass through the specimen and are focused onto an electron-sensitive detector to form a 2D cross-sectional image of the specimen. In SEM, low-voltage electrons are emitted to the surface of the specimen in a raster-scan manner, and secondary or back-scattered electrons are detected, creating a 3D appearance of the surface of the specimen.
To obtain true 3D information using TEM, tomography-based techniques (Hoppe, 1981; Baumeister, 2002) tilt an ultrathin tissue section, i.e., approximately 1 µm thick tissue, to obtain high-resolution images of macromolecules or
organelles. The TEM camera array (TEMCA) (Bock et al., 2011) or TEMCA2 (Zheng et al., 2018) are other TEM-based techniques that use serial sectioning, 40 to 90 nm thick tissue cut by an ultramicrotome, to image individual thin sections at high resolutions and acquisition rates. Acquiring 3D EM datasets using SEM includes serial block-face scanning electron microscopy (SBEM) (Denk and Horstmann, 2004), focused ion beam-scanning EM (FIB-SEM) (Knott et al., 2008), and automated tape-collecting ultramicrotomy with SEM (ATUM) (Hayworth et al., 2006). In SBEM, an ultramicrotome cuts the surface of the tissue inside an SEM chamber, and in FIB-SEM, a focused ion beam mills the surface. In ATUM, thin tissue sections are cut by an ultramicrotome, collected on a support tape automatically, and imaged with an SEM.
None of the currently available 3D TEM- and SEM-based techniques can be considered the ultimate approach for 3D EM imaging. Instead, each method has tradeoffs in size and resolution and deciding which method is the most
appropriate is relative to the questions under investigation. For example, FIB-SEM offers the highest z-resolution of 5 nm, but the tissue size is limited to 50–100 µm per side. This can be compared to ATUM and SBEM diamond-knife sectioning that offers the z-resolution of 20-30 nm but allows for larger tissue sizes; these are per side 0.5 mm for SBEM and larger than 2 mm for ATUM (Kubota, Sohn and
In this thesis, we used SBEM imaging as the method of choice to acquire EM volumes of the brain tissue ultrastructures. In the following sections, we briefly describe SEM as the backbone of SBEM and then continue with the SBEM imaging technique.
2.2.1 Scanning electron microscopy
Electrons are emitted from an electron gun (thermionic or field emission), typically accelerated by low voltages of 0.3 to 4 kV in SEM. As shown in Figure 2a, two or three electromagnetic condenser lenses focus the electron beam into a fine probe that can raster the specimen surface in a selected area. Figure 2b shows that the incident electrons penetrate the specimen surface, where the penetration depth depends on the energy of the electron beam, the atomic masses of elements in the specimen, and the angle at which the electron beam hits the specimen. The electron specimen interaction produces secondary, backscattered, and Auger electrons, x-rays, and perhaps light, which various detectors can collect. Secondary electrons are formed just below the sample surface, escape the orbitals of an atom, showing the morphology and topography of the specimen surface and providing the highest resolution. Backscattered electrons approach the atom Figure 2. (a) Schematic of a scanning electron microscopy. This figure is from (User:Steff, modified by User:ARTEderivative work MarcoTolo, CC BY-SA 1.0, via Wikimedia Commons). (b) Electron specimen interaction. This figure is from
(Ponor, CC BY-SA 4.0, via Wikimedia Commons). (c) Schematic illustration of a serial block-face scanning electron microscope.
nucleus and are scattered by a large angle, re-emerging from the surface. There are not as many backscattered electrons as secondary electrons, and they form images with slightly lower resolution than secondary electron images, providing compositional or crystallographic information (Reimer, 1998).
2.2.2 Serial block-face scanning electron microscopy
Denk and Horstmann (Denk and Horstmann, 2004) were the first to introduce SBEM, which is now available commercially as the 3View® system (Gatan Inc., Pleasanton, CA, USA). SBEM essentially uses an ultramicrotome inside the
chamber of an SEM. The ultramicrotome cuts through the sample, exposing a new face of the sample. Backscattering contrast is used to visualize the heavy-metal-stained tissue, and low-vacuum conditions prevent any charging of the uncoated block face. This procedure is repeated throughout the entire sample, resulting in a stack of high-resolution, high-contrast images, where each layer can be as small as 25 to 30 nm. SBEM imaging throughput is fast, approximately 0.5 µs/pixel, and the method is relatively straightforward. Compared to the FIB-SEM technique, SBEM can image a larger area because the diamond knife can cut the entire block
surface. Compared to ATUM, the SBEM technique images the block surface located right under the SEM column so that the acquired images are minimally affected by movements (Kubota, Sohn and Kawaguchi, 2018). Compared to TEMCA, SBEM has a longer throughput time and acquires images in a smaller field of view, but TEMCA is an expensive approach and requires customized features and machines that are not yet commercially available. It is noteworthy that the SBEM imaging technique comes with several aspects that need consideration, e.g., debris can fall on the block surface, the sections can crumble at long dwell-time, or an electric charge can accumulate at the block surface (Kubota, Sohn and Kawaguchi, 2018).