thumb|Image of [[pollen grains taken on a SEM shows the characteristic depth of field of SEM micrographs]]

thumb|right|[[Manfred von Ardenne|M. von Ardenne's first SEM]]

thumb|right|SEM with opened sample chamber

thumb|right|Analog type SEM

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, followed by the Cambridge groups in the 1950s and early 1960s 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

thumb|Schottky-emitter electron source

thumb|Electron–matter interaction volume and types of signal generated

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. 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.) 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 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.

Changing magnification is achieved by adjusting the length of the scan on the specimen, with higher magnification requiring a smaller sample area, or pixel. According to Goldstein et al, "The development of the E-T detector provided the first efficient use of the rich secondary/backscattered electron signal with a large solid angle of collection, high amplifier gain, low noise, and robust, low-maintenance performance." Light is emitted when an energetic electron strikes scintillator material, which is conducted via a light guide to a photomultiplier, which provides high gain, little noise degradation, high bandwidth, at a fast response rate. According to Goldstein et al, "When the E-T detector is biased negatively, only backscattered electrons are detected. All secondary electrons are rejected, including those that are emitted from the specimen in the direction of the E-T detector within the line-of-sight solid angle for direct geometric collection. Dedicated backscattered electron detectors are designed to greatly increase the solid angle of collection." These include the Passive Scintillator Backscattered Electron Detectors, the Backscattered-to-Secondary Electron Conversion Detector, and the Solid State Diode Detector.

Environmental SEM

Conventional SEM requires samples to be imaged under vacuum, because a gas atmosphere rapidly spreads and attenuates electron beams. As a consequence, samples that produce a significant amount of vapour, e.g. wet biological samples or oil-bearing rock, must be either dried or cryogenically frozen. Processes involving phase transitions, such as the drying of adhesives or melting of alloys, liquid transport, chemical reactions, and solid-air-gas systems, in general cannot be observed with conventional high-vacuum SEM. In environmental SEM (ESEM), the chamber is evacuated of air, but water vapor is retained near its saturation pressure, and the residual pressure remains relatively high. This allows the analysis of samples containing water or other volatile substances. With ESEM, observations of living insects have been possible.

The first commercial development of the ESEM in the late 1980s allowed samples to be observed in low-pressure gaseous environments (e.g. 1–50 Torr or 0.1–6.7 kPa) and high relative humidity (up to 100%). This was made possible by the development of a secondary-electron detector capable of operating in the presence of water vapour and by the use of pressure-limiting apertures with differential pumping in the path of the electron beam to separate the vacuum region (around the gun and lenses) from the sample chamber. The first commercial ESEMs were produced by the ElectroScan Corporation in USA in 1988. ElectroScan was taken over by Philips (who later sold their electron-optics division to FEI Company) in 1996.

ESEM is especially useful for non-metallic and biological materials because coating with carbon or gold is unnecessary. Uncoated plastics and elastomers can be routinely examined, as can uncoated biological samples. This is useful because coating can be difficult to reverse, may conceal small features on the surface of the sample and may reduce the value of the results obtained. X-ray analysis is difficult with a coating of a heavy metal, so carbon coatings are routinely used in conventional SEMs, but ESEM makes it possible to perform X-ray microanalysis on uncoated non-conductive specimens; however some specific for ESEM artifacts are introduced in X-ray analysis. ESEM may be the preferred for electron microscopy of unique samples from criminal or civil actions, where forensic analysis may need to be repeated by several different experts. It is possible to study specimens in liquid with ESEM or with other liquid-phase electron microscopy methods.

Transmission SEM and Energy Loss Spectroscopy

The SEM can also be used in transmission mode by incorporating an appropriate detector below a thin specimen section. Detectors are available for bright field, dark field, as well as segmented detectors for mid-field to high angle annular dark-field. Despite the difference in instrumentation, this technique is still commonly referred to as scanning transmission electron microscopy (STEM).

Since 2016, there has been growing interest in the use of transmission-mode SEM for Electron Energy Loss Spectroscopy (EELS). Pioneered by Hitachi and the Gauvin group at McGill University, SEM-EELS elemental mapping of thin specimens is now possible, along with reduced beam damage in lithiated materials due to the lower beam energy reducing knock-on damage. In 2025, scientists from MIT demonstrated that EELS analysis of thin specimens is possible in any retrofitted SEM in the 1-20 keV energy range.

SEM in forensic science

The SEM is used often in Forensic Science for magnified analysis of microscopic things such as diatoms and gunshot residue. Because SEM is a nondestructive force on the sample, it can be used to analyze evidence without damaging it. The SEM shoots a beam of high energy electrons to the sample which bounce off of the sample without changing or destroying it. This is great when it comes to analyzing diatoms. When a person dies by drowning, they inhale the water which causes what is in the water (diatoms) to get in the blood stream, brain, kidneys, and more. These diatoms in the body can be magnified with the SEM to determine the type of diatoms which aid in understanding how and where the person died. By using the images produced by the SEM, forensic scientists can compare diatoms types to confirm the body of water a person died in.

Gunshot residue (GSR) analysis can be done with many different analytical instruments, but SEM is a common way to analyze inorganic compounds because of the way it can closely analyze the types of elements (mostly metals) through its three detectors: backscatter electron detector, secondary electron detector, and X-ray detector. GSR can be collected from the crime scene, victim, or shooter and analyzed with the SEM. This can help scientists determine proximity and or contact with the discharged firearm. However, several methods can used to get color electron microscopy images.

False color using a single detector

  • On compositional images of flat surfaces (typically BSE):

The easiest way to get color is to replace each grey level with an arbitrary color, using a color look-up table. This method is known as false color imaging and can help to distinguish phases of the sample with similar properties or composition.

  • On textured-surface images:

As an alternative to simply replacing each grey level by a color, a sample observed by an oblique beam allows researchers to create an approximative topography image (see further section "Photometric 3D rendering from a single SEM image"). Such topography can then be processed by 3D-rendering algorithms for a more natural rendering of the surface texture.

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File:Surface of a kidney stone.jpg|Surface of a kidney stone

File:Surface of a kidney stone Re-colorized SEM Image.jpg|The same after re-processing of the color from the estimated topography

File:Discoaster-side-diag-alt hg.jpg|SEM image of a diagenetically altered discoaster

File:Discoaster-side-diag-alt Re-colorized SEM Image.jpg|The same image after similar colorization

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SEM image coloring

Very often, published SEM images are artificially colored.

Coloring may be performed manually with photo-editing software, or semi-automatically with dedicated software using feature-detection or object-oriented segmentation.

Alternately, when additional information from other detectors like EDX, EBSD, ECCI or cathodoluminescence is available, it can be merged as color channel(s) to provide rich material information in a single, high-resolution image.

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File:Cobaea scandens1-4.jpg|SEM image of Cobaea scandens pollen

File:Cobaea scandens colorized SEM image.jpg| The same after semi-automatic coloring. Arbitrary colors help identifying the various elements of the structure.

File:Tradescantia tolmukakarvad ja õietolm.JPG |Colored SEM image of Tradescantia pollen and stamens

File:Gold on arsenopyrite SEM image.png |Colored SEM image of native gold and arsenopyrite crystal intergrowth

</gallery>

Color built using multiple electron detectors

In some configurations more information is gathered per pixel, often by the use of multiple detectors.

As a common example, secondary electron and backscattered electron detectors are superimposed and a color is assigned to each of the images captured by each detector, with a result of a combined color image where colors are related to the density of the components. This method is known as density-dependent color SEM (DDC-SEM). Micrographs produced by DDC-SEM retain topographical information, which is better captured by the secondary electrons detector and combine it to the information about density, obtained by the backscattered electron detector.

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File:DDC-SEM of calcified particle in cardiac tissue - BW - 1.jpg|DDC-SEM of calcified particle in cardiac tissue - Signal 1: SE|alt=DDC-SEM of calcified particle in cardiac tissue - Signal 1 : SE

File:DDC-SEM_of_calcified_particle_in_cardiac_tissue_-_BW_-_2.jpg| Signal 2: BSE|alt=Signal 2 : BSE

File:DDC-SEM of calcified particle in cardiac tissue - orange.jpg|Colorized image obtained from the two previous. Density-dependent color scanning electron micrograph SEM (DDC-SEM) of cardiovascular calcification, showing in orange a calcium phosphate spherical particle (denser material) and, in green, the extracellular matrix (less dense material)

File:Cardiovascular calcification - Sergio Bertazzo.tif|Same work with a larger view, part of a study on human cardiovascular tissue calcification

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Analytical signals based on generated photons

Measurement of the energy of photons emitted from the specimen is a common method to get analytical capabilities. Examples are the energy-dispersive X-ray spectroscopy (EDS) detectors used in elemental analysis and the error on the Ra roughness value calculated is less than 0.5%.

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Photometric 3D SEM reconstruction from a four-quadrant detector by "shape from shading"

This method typically uses a four-quadrant BSE detector (alternatively for one manufacturer, a 3-segment detector). The microscope produces four images of the same specimen at the same time, so no tilt of the sample is required. The method gives metrological 3D dimensions as far as the slope of the specimen remains reasonable.

Other approaches use more sophisticated (and sometimes GPU-intensive) methods like the optimal estimation algorithm and offer much better results at the cost of high demands on computing power.

In all instances, this approach works by integration of the slope, so vertical slopes and overhangs are ignored; for instance, if an entire sphere lies on a flat, little more than the upper hemisphere is seen emerging above the flat, resulting in wrong altitude of the sphere apex. The prominence of this effect depends on the angle of the BSE detectors with respect to the sample, but these detectors are usually situated around (and close to) the electron beam, so this effect is very common.

Photometric 3D rendering from a single SEM image

This method requires an SEM image obtained in oblique low angle lighting. The grey-level is then interpreted as the slope, and the slope integrated to restore the specimen topography. This method is interesting for visual enhancement and the detection of the shape and position of objects; however the vertical heights cannot usually be calibrated, contrary to other methods such as photogrammetry.

  • Multi-Resolution reconstruction using single 2D File: High-quality 3D imaging may be an ultimate solution for revealing the complexities of any porous media, but acquiring them is costly and time-consuming. High-quality 2D SEM images, on the other hand, are widely available. Recently, a novel three-step, multiscale, multiresolution reconstruction method is presented that directly uses 2D images in order to develop 3D models. This method, based on a Shannon Entropy and conditional simulation, can be used for most of the available stationary materials and can build various stochastic 3D models just using a few thin sections.
  • Ion-abrasion SEM (IA-SEM) is a method of nanoscale 3D imaging that uses a focused beam of gallium to repeatedly abrade the specimen surface 20 nanometres at a time. Each exposed surface is then scanned to compile a 3D image.

Applications of 3D SEM

One possible application is measuring the roughness of ice crystals. This method can combine variable-pressure environmental SEM and the 3D capabilities of the SEM to measure roughness on individual ice crystal facets, convert it into a computer model and run further statistical analysis on the model. Other measurements include fractal dimension, examining fracture surface of metals, characterization of materials, corrosion measurement, and dimensional measurements at the nano scale (step height, volume, angle, flatness, bearing ratio, coplanarity, etc.).

SEM is also used by art conservationists to discern threats to paintings' surface stability due to aging, such as the formations of complexes of zinc ions with fatty acids. Forensic scientists use SEM to detect art forgeries.

The following are examples of images taken using an SEM.

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File:Soybean cyst nematode and egg SEM.jpg|Colored SEM image of soybean cyst nematode and egg. The artificial coloring makes the image easier for non-specialists to view and understand the structures and surfaces revealed in micrographs.

File:Krilleyekils.jpg|Compound eye of Antarctic krill Euphausia superba. Arthropod eyes are a common subject in SEM micrographs due to the depth of focus that an SEM image can capture. Colored picture.

File:Antarctic krill ommatidia.jpg|Ommatidia of Antarctic krill eye, a higher magnification of the krill's eye. SEMs cover a range from light microscopy up to the magnifications available with a TEM. Colored picture.

File:SEM blood cells.jpg|SEM image of normal circulating human blood. This is an older and noisy micrograph of a common subject for SEM micrographs: red blood cells.

File:HederelloidSEM.jpg|SEM image of a hederelloid from the Devonian of Michigan (largest tube diameter is 0.75&nbsp;mm). The SEM is used extensively for capturing detailed images of micro and macro fossils.

File:BSEGlassInclusionSb.jpg|Backscattered electron (BSE) image of an antimony-rich region in a fragment of ancient glass. Museums use SEMs for studying valuable artifacts in a nondestructive manner.

File:SEGlassCorrosion.jpg|SEM image of the corrosion layer on the surface of an ancient glass fragment; note the laminar structure of the corrosion layer.

File:Photoresist SEM micrograph.JPG|SEM image of a photoresist layer used in semiconductor manufacturing taken on a field emission SEM. These SEMs are important in the semiconductor industry for their high-resolution capabilities.

File:Surface of a kidney stone.jpg|SEM image of the surface of a kidney stone showing tetragonal crystals of Weddellite (calcium oxalate dihydrate) emerging from the amorphous central part of the stone. Horizontal length of the picture represents 0.5&nbsp;mm of the figured original.

File:LightLTSEM.jpg|Two images of the same depth hoar snow crystal, viewed through a light microscope (left) and as an SEM image (right). Note how the SEM image allows for clear perception of the fine structure details which are hard to fully make out in the light microscope image.

File:Onion flake. Cells. SEM-BSE.jpg|Epidermal cells from the inner surface of an onion flake. Beneath the shagreen-like cell walls one can see nuclei and small organelles floating in the cytoplasm. This BSE-image of a lanthanoid-stained sample was taken without prior fixation, dehydration, or sputtering.

File:SEM-stomata-UIowa.tif|SEM image of stomata on the lower surface of a leaf

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See also

References

;General

  • HowStuffWorks – How Scanning Electron Microscopes Work
  • Learn to use an SEM – An online learning environment for people wanting to use an SEM. Provided by Microscopy Australia
  • Virtual SEM – sparkler an interactive simulation of a scanning electron microscope (SEM)
  • Multichannel color SEM imaging and with BSE
  • Video on the scanning electron microscope, Karlsruhe University of Applied Sciences
  • Animations and explanations on various types of microscopes including electron microscopes (Université Paris Sud)

;History

  • Environmental Scanning Electron Microscope (ESEM) history

;Images

  • Rippel Electron Microscope Facility Many dozens of (mostly biological) SEM images from Dartmouth College.
  • Lanthanoid staining SEM images from Research Institute of Eye Diseases, Moscow.