Materials to be viewed under an 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 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 so rapidly, to liquid nitrogen or even liquid helium temperatures, 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.[citation needed]
- Dehydration – freeze drying, or replacement of water with organic solvents such as ethanol or acetone, followed by critical point drying or infiltration with embedding resins.
- 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 epoxy propane and then infiltrated with a resin such as Araldite epoxy resin; tissues may also be embedded directly in water-miscible acrylic resin. After the resin has been polymerised (hardened) the sample is thin sectioned (ultrathin sections) and stained - it is then ready for viewing.
- Embedding, materials - after embedding in resin, the specimen is usually ground and polished to a mirror-like finish using ultra-fine abrasives. The polishing process must be performed carefully to minimize scratches and other polishing artifacts that reduce image quality.
- Sectioning – produces thin slices of specimen, semitransparent to electrons. These can be cut on an ultramicrotome with a diamond knife to produce ultrathin slices 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 are 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.
- Freeze-fracture or freeze-etch – a preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view. The fresh tissue or cell suspension is frozen rapidly (cryofixed), then fractured by simply breaking or by using a microtome 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) is then shadowed with evaporated platinum or gold at an average angle of 45° in a high vacuum evaporator. A second coat of carbon, evaporated perpendicular to the average surface plane is often performed to improve 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 from residual chemicals, carefully fished up on fine grids, dried then viewed in the TEM.
- 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 SEM analysis of materials that are difficult to prepare using mechanical polishing.
- Conductive Coating – an ultrathin coating of electrically-conducting material, deposited either by high vacuum evaporation or by low vacuum sputter coating of the sample. This is done to prevent the accumulation of static electric fields at the specimen due to the electron irradiation required during imaging. Such coatings include gold, gold/palladium, platinum, tungsten, graphite etc. and are especially important for the study of specimens with the scanning electron microscope. Another reason for coating, even when there is more than enough conductivity, is to improve contrast, a situation more common with the operation of a FESEM (field emission SEM).
Disadvantages
Pictured: First degree filter setae with V-shaped second degree setae pointing towards the inside of the feeding basket. The purple ball is 1 µm in diameter.
Electron microscopes are expensive to build and maintain, but the capital and running costs of confocal light microscope systems now overlaps with those of basic electron microscopes. They are dynamic rather than static in their operation, requiring extremely stable high-voltage supplies, extremely stable currents to each electromagnetic coil/lens, continuously-pumped high- or ultra-high-vacuum systems, and a cooling water supply circulation through the lenses and pumps. As they are very sensitive to vibration and external magnetic fields, microscopes designed to achieve high resolutions must be housed in stable buildings (sometimes underground) with special services such as magnetic field cancelling systems. Some desktop low voltage electron microscopes have TEM capabilities at very low voltages (around 5 kV) without stringent voltage supply, lens coil current, cooling water or vibration isolation requirements and as such are much less expensive to buy and far easier to install and maintain, but do not have the same ultra-high (atomic scale) resolution capabilities as the larger instruments.
The samples largely have to be viewed in vacuum, as the molecules that make up air would scatter the electrons. One exception is the environmental scanning electron microscope, which allows hydrated samples to be viewed in a low-pressure (up to 20 Torr/2.7 kPa), wet environment.
Scanning electron microscopes usually image conductive or semi-conductive materials best. Non-conductive materials can be imaged by an environmental scanning electron microscope. A common preparation technique is to coat the sample with a several-nanometer layer of conductive material, such as gold, from a sputtering machine; however, this process has the potential to disturb delicate samples.
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. It is generally believed by scientists working in the field that as results from various preparation techniques have been compared and that there is no reason that they should all produce similar artifacts, it is reasonable to believe that electron microscopy features correspond with those of living cells. In addition, higher-resolution work has been directly compared to results from X-ray crystallography, providing independent confirmation of the validity of this technique.[citation needed] Since the 1980s, analysis of cryofixed, vitrified specimens has also become increasingly used by scientists, further confirming the validity of this technique.
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