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 through Ernst Ruska and colleagues in the 30’s electron microscopy greatly contributed to the structural analysis of cells, organelles, viruses and proteins. Electron microscopes have a greater resolving power than light optical microscopes, because electrons have wavelengths about 100,000 times shorter than visible light (photons), and can achieve better than 50 pm resolution and magnifications of up to about 10,000,000x, whereas ordinary light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x.
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 CCD (charge-coupled device) camera.
Over the past decade, cryo-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 electron microscopic imaging modalities when applied to samples embedded in vitreous ice. Three major branches of cryo-electron microscopy 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.
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. like the ribosome or the 26S proteasome) at a routine resolution of less than 10 Å. Sample size range: 5-50 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. Maximum sample thickness: a few 100 nm.