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History
The first SEM image was obtained by Max Knoll, who in 1935 obtained an image of silicon steel showing electron channeling contrast. Further pioneering work on the physical principles of the SEM and beam specimen interactions was performed by Manfred von Ardenne in 1937, who produced a British patent but never made a practical instrument. The SEM was further developed by Professor Sir Charles Oatley and his postgraduate student Gary Stewart and was first marketed in 1965 by the Cambridge Instrument Company as the “Stereoscan”. The first instrument was delivered to DuPont.
Scanning process and image formation
In a typical SEM, an electron beam is thermionically emitted from an electron gun fitted with a tungsten filament cathode. Tungsten is normally used in thermionic electron guns because it has the highest melting point and lowest vapour pressure of all metals, thereby allowing it to be heated for electron emission, and because of its low cost. Other types of electron emitters include lanthanum hexaboride (LaB6) cathodes, which can be used in a standard tungsten filament SEM if the vacuum system is upgraded and field emission guns (FEG), which may be of the cold-cathode type using tungsten single crystal emitters or the thermally-assisted Schottky type, using emitters of zirconium oxide.
The electron beam, which typically has an energy ranging from a few hundred eV to 40 keV, is focused by one or two condenser lenses to a spot about 0.4 nm to 5 nm in diameter. The beam passes through pairs of scanning coils or pairs of deflector plates in the electron column, typically in the final lens, which deflect the beam in the x and y axes so that it scans in a raster fashion over a rectangular area of the sample surface , price computing scale .
When the primary electron beam interacts with the sample, the electrons lose energy by repeated random scattering and absorption within a teardrop-shaped volume of the specimen known as the interaction volume, which extends from less than 100 nm to around 5 m into the surface. The size of the interaction volume depends on the electron’s landing energy, the atomic number of the specimen and the specimen’s density. The energy exchange between the electron beam and the sample results in the reflection of high-energy electrons by elastic scattering, emission of secondary electrons by inelastic scattering and the emission of electromagnetic radiation, each of which can be detected by specialized detectors. The beam current absorbed by the specimen can also be detected and used to create images of the distribution of specimen current. Electronic amplifiers of various types are used to amplify the signals which are displayed as variations in brightness on a cathode ray tube. The raster scanning of the CRT display is synchronised with that of the beam on the specimen in the microscope, and the resulting image is therefore a distribution map of the intensity of the signal being emitted from the scanned area of the specimen. The image may be captured by photography from a high resolution cathode ray tube, but in modern machines is digitally captured and displayed on a computer monitor and saved to a computer’s hard disc , currency counter .
Magnification
Magnification in a SEM can be controlled over a range of up to 6 orders of magnitude from about x25 to x 250,000 and exceptionally to 2 million times in the Hitachi S-5500 in-lens Field Emission SEM, imaging a specimen area about 60nm wide with resolution up to 0.4 nm. Unlike optical and transmission electron microscopes, image magnification in the SEM is not a function of the power of the objective lens. SEMs may have condenser and objective lenses, but their function is to focus the beam to a spot, and not to image the specimen. Provided the electron gun can generate a beam with sufficiently small diameter, a SEM could in principle work entirely without condenser or objective lenses, although it might not be very versatile or achieve very high resolution. In a SEM, as in scanning probe microscopy, magnification results from the ratio of the dimensions of the raster on the specimen and the raster on the display device. Assuming that the display screen has a fixed size, higher magnification results from reducing the size of the raster on the specimen, and vice versa. Magnification is therefore controlled by the current supplied to the x,y scanning coils, and not by objective lens power.
Sample preparation
An insect coated in gold, having been prepared for viewing with a scanning electron microscope.
All samples must also be of an appropriate size to fit in the specimen chamber and are generally mounted rigidly on a specimen holder called a specimen stub. Several models of SEM can examine any part of a 6-inch (15 cm) semiconductor wafer, and some can tilt an object of that size to 45 degrees.
For conventional imaging in the SEM, specimens must be electrically conductive, at least at the surface, and electrically grounded to prevent the accumulation of electrostatic charge at the surface. Metal objects require little special preparation for SEM except for cleaning and mounting on a specimen stub. Nonconductive specimens tend to charge when scanned by the electron beam, and especially in secondary electron imaging mode, this causes scanning faults and other image artifacts. They are therefore usually coated with an ultrathin coating of electrically-conducting material, commonly gold, deposited on the sample either by low vacuum sputter coating or by high vacuum evaporation. Conductive materials in current use for specimen coating include gold, gold/palladium alloy, platinum, osmium, iridium, tungsten, chromium and graphite. Coating prevents the accumulation of static electric charge on the specimen during electron irradiation.
Two important reasons for coating, even when there is more than enough specimen conductivity to prevent charging, are to maximise signal and improve spatial resolution, especially with samples of low atomic number (Z). Broadly, signal increases with atomic number, especially for backscattered electron imaging. The improvement in resolution arises because in low-Z materials such as carbon, the electron beam can penetrate several micrometres below the surface, generating signals from an interaction volume much larger than the beam diameter and reducing spatial resolution. Coating with a high-Z material such as gold maximises secondary electron yield from within a surface layer a few nm thick, and suppresses secondary electrons generated at greater depths, so that the signal is predominantly derived from locations closer to the beam and closer to the specimen surface than would be the case in an uncoated, low-Z material. These effects are particularly, but not exclusively, relevant to biological samples.
An alternative to coating for some biological samples is to increase the bulk conductivity of the material by impregnation with osmium using variants of the OTO process. Nonconducting specimens may be imaged uncoated using specialized SEM instrumentation such as the “Environmental SEM” (ESEM) or field emission gun (FEG) SEMs operated at low voltage. Environmental SEM instruments place the specimen in a relatively high pressure chamber where the working distance is short and the electron optical column is differentially pumped to keep vacuum adequately low at the electron gun. The high pressure region around the sample in the ESEM neutralizes charge and provides an amplification of the secondary electron signal. Low voltage (LV) SEM of non-conducting specimens can be operationally difficult to accomplish in a conventional SEM and is typically a research application for specimens that are sensitive to the process of applying conductive coatings. LV-SEM is typically conducted in an FEG-SEM because the FEG is capable of producing high primary electron brightness even at low accelerating potentials. Operating conditions must be adjusted such that the local space charge is at or near neutral with adequate low voltage secondary electrons being available to neutralize any positively charged surface sites. This requires that the primary electron beam’s potential and current be tuned to the characteristics of the sample specimen.
Embedding in a resin with further polishing to a mirror-like finish can be used for both biological and materials specimens when imaging in backscattered electrons or when doing quantitative X-ray microanalysis.
Biological samples
For SEM, a specimen is normally required to be completely dry, since the specimen chamber is at high vacuum. Hard, dry materials such as wood, bone, feathers, dried insects or shells can be examined with little further treatment, but living cells and tissues and whole, soft-bodied organisms usually require chemical fixation to preserve and stabilize their structure. Fixation is usually performed by incubation in a solution of a buffered chemical fixative, such as glutaraldehyde, sometimes in combination with formaldehyde and other fixatives, and optionally followed by postfixation with osmium tetroxide. The fixed tissue is then dehydrated. Because air-drying causes collapse and shrinkage, this is commonly achieved by critical point drying, which involves replacement of water in the cells with organic solvents such as ethanol or acetone, and replacement of these solvents in turn with a transitional fluid such as liquid carbon dioxide at high pressure. The carbon dioxide is finally removed while in a supercritical state, so that no gas-liquid interface is present within the sample during drying. The dry specimen is usually mounted on a specimen stub using an adhesive such as epoxy resin or electrically-conductive double-sided adhesive tape,…
