A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that contain information about the surface topography and composition. The electron beam is scanned in a raster scan pattern, and the position of the beam is combined with the intensity of the detected signal to produce an image. In the most common SEM mode, secondary electrons emitted by atoms excited by the electron beam are detected using a secondary electron detector (Everhart–Thornley detector). The number of secondary electrons that can be detected, and thus the signal intensity, depends, among other things, on specimen topography. Some SEMs can achieve resolutions better than 1 nanometer.
Specimens are observed in high vacuum in a conventional SEM, or in low vacuum or wet conditions in a variable pressure or environmental SEM, and at a wide range of cryogenic or elevated temperatures with specialized instruments.
History An account of the early history of scanning electron microscopy has been presented by McMullan. Although Max Knoll produced a photo with a 50 mm object-field-width showing channeling contrast by the use of an electron beam scanner, it was Manfred von Ardenne who in 1937 invented a microscope with high resolution by scanning a very small raster with a demagnified and finely focused electron beam. In the same year, Cecil E. Hall also completed the construction of the first emission microscope in North America, just two years after being tasked by his supervisor, E. F. Burton at the University of Toronto. Ardenne applied scanning of the electron beam in an attempt to surpass the resolution of the transmission electron microscope (TEM), as well as to mitigate substantial problems with chromatic aberration inherent to real imaging in the TEM. He further discussed the various detection modes, possibilities and theory of SEM, together with the construction of the first high resolution SEM. Further work was reported by Zworykin's group,Zworykin VA, Hillier J, Snyder RL (1942) A scanning electron microscope. ASTM Bull 117, 15–23. followed by the Cambridge groups in the 1950s and early 1960sOatley CW, Nixon WC, Pease RFW (1965) Scanning electron microscopy. Adv Electronics Electron Phys 21, 181–247.Wells OC (1957) The construction of a scanning electron microscope and its application to the study of fibres. PhD Dissertation, Cambridge University. headed by Charles Oatley, all of which finally led to the marketing of the first commercial instrument by Cambridge Scientific Instrument Company as the "Stereoscan" in 1965, which was delivered to DuPont.
Principles and capacities
The signals used by an SEM to make an image result from interactions between the electron beam and atoms at various depths within the sample. Various types of signals are produced including secondary electrons (SE), reflected or back-scattered electrons (BSE), characteristic X-rays and light (cathodoluminescence) (CL), absorbed current (specimen current) and transmitted electrons. Secondary electron imaging and back-scattered electron detectors are standard approaches in an SEM, but additional detectors may be used to capture additional signals. For instance, emitted X-rays can be detected by energy dispersive X-ray spectrometry.
Secondary electrons have very low energies on the order of 50 eV, which limits their mean free path in solid matter. Consequently, SEs can only escape from the top few nanometers of the surface of a sample. The signal from secondary electrons tends to be highly localized at the point of impact of the primary electron beam, making it possible to collect images of the sample surface with a resolution of below 1 nm. Back-scattered electrons (BSE) are beam electrons that are reflected from the sample by elastic scattering. Since they have much higher energy than SEs, they emerge from deeper locations within the specimen and, consequently, the resolution of BSE images is less than SE images. However, BSE are often used in analytical SEM, along with the spectra made from the characteristic X-rays, because the intensity of the BSE signal is strongly related to the atomic number (Z) of the specimen. BSE images can provide information about the distribution, but not the identity, of different elements in the sample. In samples predominantly composed of light elements, such as biological specimens, BSE imaging can image colloidal gold immuno-labels of 5 or 10 nm diameter, which would otherwise be difficult or impossible to detect in secondary electron images. Characteristic X-rays are emitted when the electron beam removes an inner shell electron from the sample, causing a higher-energy electron to fill the shell and release energy. The energy or wavelength of these characteristic X-rays can be measured by Energy-dispersive X-ray spectroscopy or Wavelength-dispersive X-ray spectroscopy and used to identify and measure the abundance of elements in the sample and map their distribution.
Due to the very narrow electron beam, SEM micrographs have a large depth of field yielding a characteristic three-dimensional appearance useful for understanding the surface structure of a sample. This is exemplified by the micrograph of pollen shown above. A wide range of magnifications is possible, from about 10 times (about equivalent to that of a powerful hand-lens) to more than 500,000 times, about 250 times the magnification limit of the best light microscopes.
Sample preparation
SEM samples have to be small enough to fit on the sample stage, secured with a conductive paint. The relatively low vacuum (0.076 to 20 torr) of Variable-Pressure and Environmental SEM obviates the need for cleaning, even those with water or oily films, allowing observation in a natural state. Preparation is identical to the steps used in light microscopy polished samples. SEM techniques can be used to examine integrated circuit condition and attachment points. Foreshortening is achieved by tilting any specimen. Sputter coating is used to apply a thin coating of metal, or metal alloy, to nonconductive specimens. Conductive materials in current use for specimen coating include gold, gold/palladium alloy, platinum, iridium, tungsten, chromium, osmium.. 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 staining method (O-osmium tetroxide, T-thiocarbohydrazide, O-osmium).
Biological samples Since the SEM specimen chamber is under high vacuum, a SEM specimen must be completely dry or cryogenically cooled. Hard, dry materials such as wood, bone, feathers, dried insects, or shells (including egg shells) can be examined with little further treatment, but living cells and tissues and whole, soft-bodied organisms 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 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 by critical point drying. 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, and sputter-coated with gold or gold/palladium alloy before examination in the microscope. Samples may be sectioned (with a microtome) if information about the organism's internal ultrastructure is to be exposed for imaging.
If the SEM is equipped with a cold stage for cryo microscopy, cryofixation may be used and low-temperature scanning electron microscopy performed on the cryogenically fixed specimens. Cryo-fixed specimens may be cryo-fractured under vacuum in a special apparatus to reveal internal structure, sputter-coated and transferred onto the SEM cryo-stage while still frozen. Low-temperature scanning electron microscopy (LT-SEM) is also applicable to the imaging of temperature-sensitive materials such as ice and fats.
Freeze-fracturing, freeze-etch or freeze-and-break is a preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view. The preparation method reveals the proteins embedded in the lipid bilayer.
Materials According to Goldstein et al, "There are many ways to section the samples, including blade sawing, wire sawing, abrasive cutting, fracturing (best for more brittle materials), shearing, spark erosion, and microtomy. The surface to be prepared for microstructural analysis is polished using a graded sequence of abrasive materials. Ceramic and geological samples, like metals, may require etching to permit microstructural features to be imaged or analyzed in the SEM." However, the electron beam path from the specimen surface to ground must remain unbroken to ensure the specimen does not act like an electron mirror when sufficient charge builds up equal to that of the incident beam, which is referred to as "charging." According to Goldstein et al, "The best and simplest way to overcome charging problems is to deposit a thin metal layer on the surface of the sample." This is achieved via vacuum evaporation coating or sputter coating. For elements with atomic numbers 8 through 20, only carbon, aluminum and chromium are suitable coating materials.
FIB is analogous to SEM, but with the use of positively charged ions instead of electrons. Besides imaging, FIB can be used for precision cutting, etching, or depositing conductive materials on the sample surface.
Scanning process and image formation
The SEM consists of an electron column, with the electron gun, electron lenses and vacuum pumps, plus a control console, with viewing screen and electron beam controls. The electron gun generates and accelerates electrons between 0.1 to 30 keV. Electron lenses focus the electron beam to less than 10 nm at the sample, where it interacts with the specimen to a depth of about 1 micrometre, generating signals used for point by point imaging. Deflection coils sweep the electron beam across the specimen in a raster scan fashion, with the backscattered and secondary electrons collected by the electron detector, forming an image that can be processed and displayed digitally. Sharpness and feature visibility are controlled by electron probe size, electron probe current, electron probe convergence angle and electron beam accelerating voltage. According to Goldstein et al, "Most older SEMs use tungsten or LaB6 thermionic emitters, but, increasingly, new microscopes are equipped with cold, thermal, or Schottly field emission sources because these provide enhanced performance, reliability, and lifetime."
Magnification Magnification in an SEM can be controlled over a range of about 6 orders of magnitude from about 10 to 3,000,000 times. Changing magnification is achieved by adjusting the length of the scan on the specimen, with higher magnification requiring a smaller sample area, or pixel.
Detection of secondary electrons