Electron microscope

Reproduction of an early electron microscope constructed by Ernst Ruska

Many developments laid the groundwork of the electron optics used in microscopes.[1] One significant step was the work of Hertz in 1883[2] who made a cathode-ray tube with electrostatic and magnetic deflection, demonstrating manipulation of the direction of an electron beam. Others were focusing of the electrons by an axial magnetic field by Emil Wiechert in 1899,[3] improved oxide-coated cathodes which produced more electrons by Arthur Wehnelt in 1905[4] and the development of the electromagnetic lens in 1926 by Hans Busch.[5] According to Dennis Gabor, the physicist Leó Szilárd tried in 1928 to convince him to build an electron microscope, for which Szilárd had filed a patent.[6]

To this day the issue of who invented the transmission electron microscope is controversial.[7][8][9][10] In 1928, at the Technical University of Berlin, Adolf Matthias (Professor of High Voltage Technology and Electrical Installations) appointed Max Knoll to lead a team of researchers to advance research on electron beams and cathode-ray oscilloscopes. The team consisted of several PhD students including Ernst Ruska. In 1931, Max Knoll and Ernst Ruska[11][12] successfully generated magnified images of mesh grids placed over an anode aperture. The device, a replicate of which is shown in the figure, used two magnetic lenses to achieve higher magnifications, the first electron microscope. (Max Knoll died in 1969, so did not receive a share of the Nobel Prize in 1986.)

Apparently independent of this effort was work at Siemens-Schuckert by Reinhold Rüdenberg. According to patent law (U.S. Patent No. 2058914[13] and 2070318,[14] both filed in 1932), he is the inventor of the electron microscope, but it is not clear when he had a working instrument. He stated in a very brief article in 1932[15] that Siemens had been working on this for some years before the patents were filed in 1932, claiming that his effort was parallel to the university development. He died in 1961, so similar to Max Knoll, was not eligible for a share of the Nobel Prize.

In the following year, 1933, Ruska and Knoll built the first electron microscope that exceeded the resolution attainable with an optical (light) microscope.[16] Four years later, in 1937, Siemens financed the work of Ernst Ruska and Bodo von Borries, and employed Helmut Ruska, Ernst's brother, to develop applications for the microscope, especially with biological specimens.[16][17] Also in 1937, Manfred von Ardenne pioneered the scanning electron microscope.[18] Siemens produced the first commercial electron microscope in 1938.[19] The first North American electron microscopes were constructed in the 1930s, at the Washington State University by Anderson and Fitzsimmons [20] and at the University of Toronto by Eli Franklin Burton and students Cecil Hall, James Hillier, and Albert Prebus. Siemens produced a transmission electron microscope (TEM) in 1939.[21] Although current transmission electron microscopes are capable of two million-power magnification, as scientific instruments they remain similar but with improved optics.

Diagram illustrating the phenomena resulting from the interaction of energetic electrons with matter

In a typical electron gun, individual electrons, which have an elementary charge ${\displaystyle e}$ (about ${\displaystyle -1.6\times 10^{-19}}$ coulombs) and a mass ${\displaystyle m}$ (about ${\displaystyle 9.1\times 10^{-31}}$ kg), with a potential of ${\displaystyle V}$ volts, have an energy amount of ${\displaystyle e\cdot V}$ joules. The wavelength is[22]

${\displaystyle \lambda ={\frac {hc}{\sqrt {eV(2mc^{2}+eV)}}}}$,

where ${\displaystyle c}$ is the speed of light in vacuum (about ${\displaystyle c=3\times 10^{8}}$ m/s). See electron diffraction for a full explanation.

An insect coated in gold for viewing with a scanning electron microscope

Materials to be viewed in a transmission electron microscope may require processing to produce a suitable sample. The technique required varies depending on the specimen and the analysis required:

• Chemical fixation – for biological specimens this aims to stabilize the specimen's mobile macromolecular structure by chemical crosslinking of proteins with aldehydes such as formaldehyde and glutaraldehyde, and lipids with osmium tetroxide.
• Cryofixation – freezing a specimen rapidly in liquid ethane, so that the water forms vitreous (non-crystalline) ice. This preserves the specimen in a snapshot of its solution state. An entire field called cryo-electron microscopy has branched from this technique. With the development of cryo-electron microscopy of vitreous sections (CEMOVIS), it is now possible to observe samples from virtually any biological specimen close to its native state.
• Dehydration – replacement of water with organic solvents such as ethanol or acetone, followed by critical point drying or infiltration with embedding resins. See also freeze drying.
• Embedding, biological specimens – after dehydration, tissue for observation in the transmission electron microscope is embedded so it can be sectioned ready for viewing. To do this the tissue is passed through a 'transition solvent' such as propylene oxide (epoxypropane) or acetone and then infiltrated with an epoxy resin such as Araldite, Epon, or Durcupan;[28] tissues may also be embedded directly in water-miscible acrylic resin. After the resin has been polymerized (hardened) the sample is thin sectioned (ultrathin sections) and stained.
• Embedding, materials – after embedding in resin, the specimen is usually ground and polished to a mirror-like finish using ultra-fine abrasives.
• Freeze-fracture or freeze-etch – a preparation method[29][30][31] particularly useful for examining lipid membranes and their incorporated proteins in "face on" view.[32][33][34]
  

  Freeze-fracturing helps to peel open membranes to allow visualization of what is inside
  

  External face of bakers yeast membrane showing the small holes where proteins are fractured out, sometimes as small ring patterns.
  The fresh tissue or cell suspension is frozen rapidly (cryofixation), then fractured by breaking[35] (or by using a microtome)[34] while maintained at liquid nitrogen temperature. The cold fractured surface (sometimes "etched" by increasing the temperature to about −100 °C for several minutes to let some ice sublime)[34] is then shadowed with evaporated platinum or gold at an average angle of 45° in a high vacuum evaporator. The second coat of carbon, evaporated perpendicular to the average surface plane is often performed to improve the stability of the replica coating. The specimen is returned to room temperature and pressure, then the extremely fragile "pre-shadowed" metal replica of the fracture surface is released from the underlying biological material by careful chemical digestion with acids, hypochlorite solution or SDS detergent. The still-floating replica is thoroughly washed free from residual chemicals, carefully fished up on fine grids, dried then viewed in the TEM.
• Freeze-fracture replica immunogold labeling (FRIL) – the freeze-fracture method has been modified to allow the identification of the components of the fracture face by immunogold labeling. Instead of removing all the underlying tissue of the thawed replica as the final step before viewing in the microscope the tissue thickness is minimized during or after the fracture process. The thin layer of tissue remains bound to the metal replica so it can be immunogold labeled with antibodies to the structures of choice. The thin layer of the original specimen on the replica with gold attached allows the identification of structures in the fracture plane.[36] There are also related methods which label the surface of etched cells[37] and other replica labeling variations.[38]
• Ion beam milling – thins samples until they are transparent to electrons by firing ions (typically argon) at the surface from an angle and sputtering material from the surface. A subclass of this is focused ion beam milling, where gallium ions are used to produce an electron transparent membrane in a specific region of the sample, for example through a device within a microprocessor. Ion beam milling may also be used for cross-section polishing prior to analysis of materials that are difficult to prepare using mechanical polishing.
• Negative stain – suspensions containing nanoparticles or fine biological material (such as viruses and bacteria) are briefly mixed with a dilute solution of an electron-opaque solution such as ammonium molybdate, uranyl acetate (or formate), or phosphotungstic acid. This mixture is applied to a suitably coated EM grid, blotted, then allowed to dry. Viewing of this preparation in the TEM should be carried out without delay for best results. The method is important in microbiology for fast but crude morphological identification, but can also be used as the basis for high-resolution 3D reconstruction using EM tomography methodology when carbon films are used for support. Negative staining is also used for observation of nanoparticles.
• Sectioning – produces thin slices of the specimen, semitransparent to electrons. These can be cut on an ultramicrotome with a glass or diamond knife to produce ultra-thin sections about 60–90 nm thick. Disposable glass knives are also used because they can be made in the lab and are much cheaper.
• Staining – uses heavy metals such as lead, uranium or tungsten to scatter imaging electrons and thus give contrast between different structures, since many (especially biological) materials are nearly "transparent" to electrons (weak phase objects). In biology, specimens can be stained "en bloc" before embedding and also later after sectioning. Typically thin sections are stained for several minutes with an aqueous or alcoholic solution of uranyl acetate followed by aqueous lead citrate.[39]

JEOL transmission and scanning electron microscope made in the mid-1970s

Electron microscopes are expensive to build and maintain. Microscopes designed to achieve high resolutions must be housed in stable buildings (sometimes underground) with special services such as magnetic field canceling systems.

The samples largely have to be viewed in vacuum, as the molecules that make up air would scatter the electrons. An exception is liquid-phase electron microscopy[40] using either a closed liquid cell or an environmental chamber, for example, in the environmental scanning electron microscope, which allows hydrated samples to be viewed in a low-pressure (up to 20 Torr or 2.7 kPa) wet environment. Various techniques for in situ electron microscopy of gaseous samples have been developed.[41]

Scanning electron microscopes operating in conventional high-vacuum mode usually image conductive specimens; therefore non-conductive materials require conductive coating (gold/palladium alloy, carbon, osmium, etc.). The low-voltage mode of modern microscopes makes possible the observation of non-conductive specimens without coating. Non-conductive materials can be imaged also by a variable pressure (or environmental) scanning electron microscope.

Small, stable specimens such as carbon nanotubes, diatom frustules and small mineral crystals (asbestos fibres, for example) require no special treatment before being examined in the electron microscope. Samples of hydrated materials, including almost all biological specimens, have to be prepared in various ways to stabilize them, reduce their thickness (ultrathin sectioning) and increase their electron optical contrast (staining). These processes may result in artifacts, but these can usually be identified by comparing the results obtained by using radically different specimen preparation methods. Since the 1980s, analysis of cryofixed, vitrified specimens has also become increasingly used by scientists, further confirming the validity of this technique.[42][43][44]

Wikimedia Commons has media related to Electron microscopes.

• An Introduction to Microscopy Archived 2013-07-19 at the Wayback Machine: resources for teachers and students
• Cell Centered Database – Electron microscopy data
• Science Aid: Electron Microscopy:u