The high-resolution electron microscope has evolved into a sophisticated instrument that is capable of routinely providing quantitative structural information on the atomic scale.
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Since the development of the electron microscope by Ernst Ruska and colleagues in the 30’s, electron microscopy has greatly contributed to the structural analysis of cells, organelles, viruses and proteins. Decades of discoveries and technological developments have been recognised by the 2017 Nobel Prize in Chemistry awarded “for developing cryo-electron microscopy for high-resolution structure determination of biomolecules in solution”.
Electron microscopes have a greater resolving power than light optical microscopes, because electrons have wavelengths about one hundred thousand times shorter than visible light (photons), and can achieve better than 50 pm resolution and magnifications of up to about ten million times magnification, whereas ordinary light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below two thousand times magnification. There are two main types of electron microscopes, the scanning electron microscope and the transmission electron microscope.
A scanning electron microscope (SEM) images a sample by scanning it with a high-energy beam of electrons in a raster scan pattern. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography, composition, and other properties such as electrical conductivity.
A transmission electron microscope (TEM) uses a technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen. The image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of photographic film, or to be detected by a sensor such as a direct electron detector.
Over the past decade, cryogenic electron microscopy (cryo-EM) has increasingly replaced the traditional methods of sample preparation for electron microscopy. It was the pioneering work of Taylor and Glaeser and of Dubochet and colleagues that paved the way for this development, which presented a quantum leap of biological electron microscopy as it enabled to obtain images of fully hydrated specimens in a close-to-native state.
The term cryo-EM refers to various transmission electron microscopic imaging modalities when applied to samples embedded in vitreous ice. Three major branches of cryo-EM are relevant in the context of molecular structural biology: electron crystallography, single particle analysis and electron tomography.
Electron crystallography offers the advantage in determining the structure of proteins forming 2D-crystals below 4Å (e.g. as shown with water transporting membrane proteins - aquaporins). Membrane proteins are particularly promising candidates for the formations of 2D-crystals. Resolutions beyond 2 Å have been obtained. Most recently, three dimensional electron crystallography of protein microcrystals (microED) has been developed and is showing promises in solving high resolutions structures of proteins forming tiny 3D-crystals (200 nm) usually not amenable for study by x-ray crystallography.
Single particle analysis is used for isolated and purified larger assemblies of multiple subunits that are often very heterogeneous, metastable and extremely hard to crystallize (e.g. the ribosome or the 26S proteasome) at a routine resolution 3–10 Å and in best cases below 2 Å. Sample size range: 5-150 nm.
Electron tomography can nowadays be used for quasi in vivo studies of non-repetitive structures, such as whole cells or for example giant molecular assemblies like the nuclear pore complex. Tomograms of organelles and cells contain an imposing amount of information at a resolution of ~4-5 nm. They are, essentially, 3D images of entire proteomes, and they should ultimately enable us to map the spatial relationships of the full complement of macromolecules in an unperturbed cellular context. Furthermore, by averaging repetitive sub-structures within tomograms, such as the ribosomes, resolutions approaching that of single-particle analysis are becoming possible. Maximum sample thickness: a few 100 nm.