An introduction to scanning electron microscope (SEM)

A scanning electron microscope (SEM) utilizes a focused beam of electrons to produce images of a sample's surface by scanning it. When the electrons interact with atoms in the sample, various signals are produced that contain information about the surface topography and composition. The electron beam follows a raster scan pattern, and an image is created by combining the position of the beam with the detected signal's intensity. In the most commonly used SEM mode, secondary electrons are detected by a secondary electron detector (Everhart–Thornley detector) after being emitted by atoms excited by the electron beam. The signal intensity, and thus the number of secondary electrons that can be detected, depends on the specimen topography and other factors. Some SEMs can achieve resolutions better than 1 nanometer.

Conventional SEMs observe specimens in high vacuum, whereas variable pressure or environmental SEMs allow for observation in low vacuum or wet conditions. Specialized instruments enable SEMs to observe specimens at a wide range of cryogenic or elevated temperatures.

SEM Principle

Scanning electron microscopy (SEM) is a powerful technique used to produce images of samples by scanning the surface with a focused beam of electrons. The SEM detects various types of signals generated by the interaction of the electron beam with atoms at different depths within the sample. The most common signal detected by SEMs is secondary electrons (SE), which are emitted from the top few nanometers of the surface of the sample. SEs produce highly localized signals that enable imaging of the sample surface with sub-nanometer resolution.

In addition to SEs, SEMs can detect back-scattered electrons (BSE), which have much higher energy than SEs and can emerge from deeper locations within the specimen. BSE images are often used in analytical SEMs, along with spectra made from characteristic X-rays. The intensity of the BSE signal is strongly related to the atomic number of the specimen, which makes BSE imaging useful for providing information about the distribution of different elements in the sample.

Characteristic X-rays are another type of signal detected by SEMs. These 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 and used to identify and measure the abundance of elements in the sample and map their distribution.

One of the advantages of SEM is its ability to produce images with a large depth of field, which provides a three-dimensional appearance of the sample surface. SEMs also offer a wide range of magnifications, from approximately 10 times to over 500,000 times, which is about 250 times the magnification limit of the best light microscopes.

SEM is a versatile tool that can be used to study a wide range of samples, including biological specimens, materials, and surfaces. Its ability to produce high-resolution images with a large depth of field makes SEM a valuable tool for understanding the surface structure of samples and for analyzing the distribution of different elements within a sample.

Sample preparation

Scanning electron microscopy (SEM) is a powerful tool used in a wide range of scientific fields to study the surface features of materials and biological specimens. To prepare specimens for SEM imaging, samples must be small enough to fit on the specimen stage and may require special preparation to increase their electrical conductivity and to stabilize them to withstand the high vacuum conditions and the high energy beam of electrons. The samples are mounted on a specimen holder or stub using a conductive adhesive.

Non-conductive specimens collect charge when scanned by the electron beam, which can cause scanning faults and image artifacts. To prevent this, specimens must be electrically conductive, at least at the surface, and electrically grounded to prevent the accumulation of electrostatic charge. Metal objects require little special preparation for SEM except for cleaning and conductively mounting to a specimen stub. Non-conducting materials are usually coated with an ultrathin coating of electrically conducting material, such as gold, platinum, or graphite, to increase conductivity. Coating with heavy metals may improve the signal/noise ratio for samples of low atomic number.

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. Non-conducting specimens may also be imaged without coating using an environmental SEM (ESEM) or low-voltage mode of SEM operation. ESEM instruments place the specimen in a relatively high-pressure chamber, which neutralizes charge and provides an amplification of the secondary electron signal.

Low-voltage SEM is typically conducted in an instrument with a field emission gun (FEG) capable of producing high primary electron brightness and small spot size even at low accelerating potentials. To prevent charging of non-conductive specimens, operating conditions must be adjusted such that the incoming beam current is equal to the sum of outgoing secondary and backscattered electron currents, which is most often met at accelerating voltages of 0.3-4 kV.

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. The main preparation techniques are not required in the environmental SEM, but some biological specimens can benefit from fixation. Overall, SEM is a versatile tool for analyzing the surface features of materials and biological specimens with high resolution and magnification.

Using SEM to test materials

In the field of microscopy, several techniques require a smooth surface for effective imaging. Back-scattered electron imaging, quantitative X-ray analysis, and X-ray mapping all necessitate the polishing of specimens to achieve ultra-smooth surfaces. In some cases, carbon-coating is applied to specimens undergoing WDS or EDS analysis.

Metals, which are conductive and provide their own pathway to ground, are generally not coated before imaging in the scanning electron microscope (SEM). Fractography is a technique that involves the study of fractured surfaces, and it can be done using a light microscope or an SEM. To prepare a fractured surface for SEM imaging, it is cut to a suitable size, cleaned of any organic residues, and mounted on a specimen holder.

In the case of integrated circuits, they may be cut using a focused ion beam (FIB) or other ion beam milling instrument for viewing in the SEM. When the SEM is incorporated into the FIB, it enables high-resolution imaging of the result of the process.

To prepare metals, geological specimens, and integrated circuits for SEM imaging, they may also be chemically polished. Special high-resolution coating techniques are required for high-magnification imaging of inorganic thin films. Overall, preparing specimens for SEM imaging involves various techniques depending on the nature of the specimen and the imaging technique being used.

SEM analysis is a powerful technique that allows for high-resolution imaging of materials, making it useful for evaluating surface features such as fractures, flaws, contaminants, or corrosion.

USING SEM ON environmental samples

SEM analysis requires the specimen to be completely dry, as the specimen chamber is under high vacuum. Dry and hard materials such as shells, feathers, wood, bone, and dried insects can be examined with minimal further treatment. However, living cells, soft-bodied organisms, and tissues require chemical fixation to stabilize their structure. Fixation is typically done by immersing the sample in a solution of buffered chemical fixative, such as glutaraldehyde, often in combination with formaldehyde and other fixatives, followed by optional postfixation with osmium tetroxide. The fixed tissue is then dehydrated using organic solvents such as ethanol or acetone, followed by critical point drying with liquid carbon dioxide. The carbon dioxide is then removed while in a supercritical state, preventing any gas-liquid interface within the sample during drying.

The dried specimen is mounted on a specimen stub using an adhesive such as epoxy resin or electrically conductive double-sided adhesive tape. Before examination in the microscope, it is sputter-coated with gold or gold/palladium alloy. Samples may be sectioned using a microtome to expose information about the internal ultrastructure of the organism.

If the SEM has a cold stage for cryo microscopy, cryofixation can be used, and low-temperature scanning electron microscopy can be performed on the cryogenically fixed specimens. Cryo-fixed specimens may be cryo-fractured under vacuum 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 are preparation methods that are useful for examining lipid membranes and the proteins they contain in a "face-on" view. This method reveals the proteins embedded in the lipid bilayer.

SEM has numerous applications in the field of biological sciences, including the identification of bacterial strains, testing vaccines, and in genetics. Additionally, SEM is a valuable tool in measuring the impact of climate change on various species, as well as discovering new species.

In forensics, SEM is a dependable technique for examining gunshot residue and analyzing paint particles and fibers at crime scenes. It is also useful for analyzing handwriting and print, and for verifying the authenticity of banknotes. Furthermore, SEM is employed in the analysis of filament bulbs found at traffic accident sites.