For biologists or students that have an interest in electron microscopy but not a lot of background in the field, large volume 3DEM imaging can be thought of as like CT or MRI imaging for cells and tissues rather than people. As in human imaging, these technologies generate sliced views through the specimen, and from sets of slices (“stacks”), 3D models and measurements of internal structures can be obtained. Advantages of these approaches are numerous, and the comparatively low cost of some instruments place them within reach of many academic and commercial research institutions.
The technologies described briefly here are labeled “large volume 3DEM” to distinguish them from the atomic-scale 3D cryo-EM imaging of molecules; cryoEM is a higher level of resolution and complexity and requires specially equipped TEM systems. They are also distinct from high voltage EM tomography, in which tilt-series of TEM images are computer reconstructed to produce virtual, very high resolution, tomographic volumes.
Not Great-Grandfather’s electron microscope...
Traditional transmission electron microscopes (TEM) feature an electron beam that is focused onto a thin (70 nm) section of tissue. The section has been stained with heavy metals that bind to membranes and proteins. Where the beam strikes metals (in cell membranes for example) the electrons are deflected and so do not contribute to the final image. Images produced in this way form a negative image of the sample slice, and can have sub-nanometer resolution. Chief difficulties with TEM for 3D imaging (ssTEM), however, are (a) that someone needs to cut continuous serial section sequences with an ultramicrotome, and then (b) stain and (c) image them. Nevertheless, a vast amount of the scientific literature is based on their use.
Assessment (manual ssTEM): Politely speaking, obtaining long sequences of serial sections by manual sectioning is “not trivial”, though semi-automated systems for TEM are now available (see below). Best for labs with a dedicated TEM person and small projects that need high resolution and few sections.
Several relatively new, highly automated electron microscope technologies have made it possible to obtain the large stacks of EM images by cutting sequentially through a sample. Instruments differ in conformation and capabilities, but share common preparative methods (en bloc heavy metal staining) and analysis techniques (stack-based reconstruction).
Serial blockface SEM imaging systems feature an in-chamber ultramicrotome mounted on – or in place of – the stage of a scanning electron microscope (SEM). In traditional biological SEM, the electrons scanning across the sample are “reflected” back to a detector (secondary electron detector) that builds up an image of the cell, or fly, or rock. In serial blockface imaging, in contrast, the electrons that penetrate the sample surface are collected after they have been ejected (back scattered electrons, BSE) as a result of interactions with the atomic nuclei of heavy metals used to stain the sample. BSE images “document” structures that lie just beneath (30-50nm) the surface of the sample block (the block face) and are very similar in appearance to a TEM micrographs. In serial blockface imaging systems, a diamond knife is used to shave a thin layer from the sample surface, exposing a fresh surface for imaging each time. Cycles of shaving and imaging the block face quickly produce large sets of sequential images. This process is computer-automated.
Assessment: Serial blockface systems are fairly robust and can be operated in labs with little prior EM experience. They also produce relatively large fields for imaging. Imaging is destructive, however, which means there are no “do-overs”.
Focused ion beam SEM imaging is another way to achieve the same kind of serial BSE imaging. In FIB-SEM, a gallium ion beam is used to “mill” or polish the sample surface face away between imaging. Compared with other techniques, FIB-SEM provides finest control over the “slice” thickness; diamond knives cut a minimum of ~30nm whereas FIB-SEM systems can polish away 5nm thicknesses if needed. System costs and complexity may make them difficult for a small lab or bio-core to adopt, and this is likewise destructive.
Assessment: Great as instruments shared with engineers and material scientists, who use them routinely. They are ideal for many questions, but not as easy to operate, maintain, and provision as serial blockface SEM systems.
Serial sectioning SEM/TEM. Automated benchtop ultramicrotome systems have been developed that produce serial sections, mounted on tape, or wafers, or coverslips. By imaging the sections in sequence – like a film strip – image stacks can be generated for 3D reconstruction. Broadly called “array tomography”, imaging occurs using a high quality SEM (as for serial blockface SEM and FIB-SEM) or a modified TEM in some cases. Unlike blockface imaging, however, sections may potentially suffer from wrinkles, compression artifact, knife marks and distortion, which must be corrected digitally after acquisition.
Assessment: this is an enormously powerful approach and is at the heart of very large connectomics studies, as well as smaller “lab-scale” studies. Best for labs with a lot of prior experience in EM, and deep pockets.