The Scanning Electron Microscope

A Small World of Huge Possibilities

It's been 50 years since Cambridge Scientific Instruments launched the first commercial scanning electron microscope (SEM) in 1965. Researchers' perception of the nanoscale world has never been the same.

An Introduction to SEMs

Traditional light microscopes cannot resolve two points closer than about 200 nm apart. In the life sciences, superresolution microscopes and certain software manipulations can edge us past this limit, but subcellular structures still defy good definition—as do those fine structural features in the materials sciences, semiconductor, and other key industries. Electrons, by contrast, have wavelengths about four to five orders of magnitude shorter than visible light and using them, electron microscopes can capture images with extraordinary detail.

SEMs, developed largely thanks to the work of von Ardenne in the 1930s, and Oatley from the 1940s into the 1960s, create surface images of bulk samples—as opposed to the thin samples used in transmission electron microscopy—by scanning an electron beam over a sample, recording the resulting echoes and electrical interactions point by point. Read on to learn about the history of scanning electron microscopy and the science behind how information and images are gathered.

Timeline of SEM Development

1926Hans Busch demonstrates that charged particles can be bent in a magnetic field as glass lenses bend visible light. 1931Ernst Ruska builds the first transmission electron microscope with resolution higher than a light microscope (~12,000x). 1933Ruska (right) and Knoll working on their transmission electron microscope (Berlin). 1935Max Knoll (shown with Ruska in 1933 image) becomes the first researcher to scan a surface with an electron beam to obtain an image. Lacking lenses, his system has a resolution of ~100 μm. 1938Manfred von Ardenne develops the first scanning transmission electron microscope, with an electron beam diameter on target of ~10 nm. His first image is of a zinc oxide crystal at 8000x magnification.
1940Von Ardenne differentiates secondary and backscattered electrons, and demonstrates that reducing the beam energy improves contrast at the expense of resolution. 1942Vladimir Zworykin (sitting), James Hillier (standing), and Richard Snyder (not pictured), working at RCA, implement a working SEM instrument with 50-nm resolving power. 1948Charles Oatley begins SEM development at Cambridge University, UK, spending some two decades on the project.
1955Invention of the diamond knife for improved ultra-microtomy and block-face imaging of thick biological specimens.
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Serial block-face imaging by in situ ultramicrotomy in an SEM. This is an iterative process in which slices (down to 15 nm thickness) from a sample block are consecutively removed with a diamond knife and the block surface imaged. The resulting stack of images can be rendered as a 3D view of the specimen. Based on earlier work by Stephen B. Leighton in 1981, this technique was implemented as shown in this video by Winfried Denk and Heinz Horstmann in 2004. Shortly afterwards commercial solutions became available.
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1960Development of the Everhart-Thornley secondary electron detector. 1960Fabian Pease in Oatley's group in Cambridge, UK, achieves SEM resolution of 10 nm. 1965Cambridge Scientific Instruments releases the first commercial SEM, the Stereoscan Mark I, here being demonstrated for Great Britain's Prince Philip (second from left).
1972Albert Crewe at the University of Chicago, US, and researchers at Hitachi develop the first practical field emission (FE)-SEM. Its brighter source allowed for higher resolution, as shown by this image of a T2 bacteriophage particle. 1985ZEISS launches the first fully digital SEM, the DSM 950. 1986Ernst Ruska wins the Nobel Prize in Physics, "for his fundamental work in electron optics, and for the design of the first electron microscope." 1988ElectroScan Corp. launches the first commercial environmental SEM, enabling "wet" life science applications. 1988Pierre Sudraud and colleagues incorporate a focused ion beam (FIB) column, pioneered by Riccardo Levi-Setti (1975), into an SEM, enabling researchers to excavate the sample surface to probe its interior.
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A traditional SEM details the structural or chemical features of a sample surface, but not what lies beneath it. In 1988, Pierre Sudraud and colleagues placed a focused-ion beam at roughly a 45° angle to an SEM electron beam, enabling the simultaneous imaging and milling of samples to obtain three-dimensional datasets. Such a configuration, called crossbeam or dualbeam, is available commercially.

The focused ion beam can be used to prepare a cross section of a sample by simply milling a trench perpendicular to the sample surface. The SEM then reveals the sub-surface structure. This example shows the layered structure of a battery terminal consisting of silver and nickel layers on top of copper.

Focused ion beam (FIB) is shown milling a spiral pattern while the SEM records its progress, demonstrating how material can be removed by the FIB and how these two modalities can work in unison.
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A focused ion beam (FIB)-SEM tomography image stack of a superconducting wire. The superconducting wires are embedded in a copper matrix. During FIB-SEM tomography, the sample is progressively sectioned by the FIB, while the SEM records the images. This results in a stack of images with volumetric information.
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1990Thermal-assisted (Schottky) FE-SEMs launched, offering superior electron current stability relative to "cold-field" emitters. 1996Bernie Breton and colleagues at the University of Cambridge operate an SEM remotely over the Internet. 2003JEOL launches the first commercial "aberration-corrected" SEM, the JSM-7700F. 2014ZEISS launches MultiSEM 505, a 61-beam (multi-beam) SEM.
Learn More Traditional SEM instruments employ a single electron beam, which is scanned over the sample to produce an image. The speed of that process, however, is limited. In 2014, a multibeam SEM comprising 61 beams operating in parallel was launched. Capable of imaging samples several times faster than a conventional instrument, the ZEISS MultiSEM 505 is being used to advance research into the detailed cellular structure and connectivity of the brain, a field called "connectomics."

SEM Technology & Signal Detection

Analogous in design to laser-scanning confocal microscopes, SEMs use electromagnetic "lenses" to focus an electron beam to a sharp point and raster-scan across the sample. Instead of recording fluorescence, however, SEMs create images by recording the interactions of the electron beam with the sample surface, which could be a ceramic material, metal, or biological specimen. These interactions can take many forms, and SEM users can install a range of detectors around the sample chamber to interrogate them.

View Diagram of SEM

  • 1

    Electron source

    Electrons are extracted from a sharp tip by means of thermal activation and/or a strong electric field. Three common types of electron sources exist: thermionic, cold field, and Schottky emitters.
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  • 2

    Beam limiting aperture

    Filters out those electrons with trajectories that are not near the optical axis. This reduces the impact of spherical aberrations.

  • 3

    Scan coils

    Used to deflect the electron beam to allow raster scanning of the sample.

  • 4

    Objective lens

    Focuses the electron beam to the smallest possible spot. The final beam spot size depends on parameters such as beam current, electron landing energy, and working distance. The beam spot size and the nature of the beam-sample interaction determine the achievable imaging resolution.

  • 5

    Sample

    Virtually any sample can be studied in an SEM. Imaging of non-conducting, outgassing, or wet samples usually involves either sample preparation steps or requires special imaging conditions in the SEM (e.g., low electron beam energies or low vacuum).
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  • 6

    Detector

    Used to detect different signals resulting from the interaction of electron beam and sample. The detector signals are recorded point by point during the scan and collated to create an image.

SEM Signals

Secondary electrons (SE)

Secondary electrons (SE) are low-energy (<50 eV) electrons resulting from the "inelastic" interaction of the primary electron beam (and backscattered electrons) with the sample, revealing sample topography (and to some extent, its electrical properties). In an Everhart-Thornley (ET) detector, the most widely used SEM detector today, the SEs hit a scintillator, which converts the electron into a photon signal. This signal then is amplified to produce an image. Today's advanced SEMs complement ET detectors with so-called "through-the-lens" or "in-lens" detectors within the SEM column to provide higher resolution images.

Backscattered electrons (BSE)

BSE reflect off the sample surface like light from a mirror. As backscattering efficiency depends on atomic number, BSE can discern differences in sample composition, such as the presence of a silver coating on a metal fiber.

Cathodoluminescence (CL)

When electrons hit luminescent materials, such as certain minerals, they produce light. A CL detector picks up those photons, producing a "live-color" image in which different materials produce distinct levels of contrast. CL is primarily used in geology—for instance, in oil and gas research and mining—but it also can be used in biology to image luminescent proteins.

X-rays (EDS/WDS)

The SEM electron beam can knock electrons in the sample out of their orbitals, producing x-rays of characteristic wavelength. Detection of these x-rays by energy-dispersive x-ray spectroscopy (EDS) or wavelength-dispersive x-ray spectroscopy (WDS) reveals a sample's elemental composition. WDS provides higher energy resolution than EDS, but is slower and more expensive. Both methods are used in museum laboratories for chemical analysis and forgery detection. EDS also has applications in forensics, such as for gunshot-residue detection.

Transmitted electrons (STEM)

When imaging very thin samples (<100 nm thick), electrons can pass through the sample. Both scattered and unscattered electrons can be detected in this way, resulting in dark-field and bright-field scanning transmission electron microscopy (STEM) images of particularly high resolution. Similar in concept to transmission EM, but imaging at lower electron voltages, STEM has both materials science and life science applications.

Backscatter diffracted electrons (EBSD)

Bragg diffraction occurs when the imaging beam strikes a crystalline material. An electron backscatter diffraction (EBSD) pattern results, which reveals key details of the crystal's underlying structure, including its phase. Among other applications, EBSD is used in solar cell development to ensure that the cells are fabricated correctly.

Correlative microscopy

The interaction of the electron beam and sample delivers many different signals that provide a wealth of information. However, many scientific problems require the combination of SEM with other complementary microscopy techniques. While SEM provides high-resolution chemical and structural information on bulk samples, researchers may want to identify regions of interest using other methods, such as light microscopy, that they then wish to subject to more detailed SEM analysis. This approach, called CLEM ("correlative light and electron microscopy") is complicated by the fact that light and electron microscopy offer such dramatically different fields of view, making it difficult to identify the same features at different size scales. Companies now offer tools to simplify the process, including sample adaptors equipped with "fiducial markers" for easy triangulation.

Correlative microscopy using light (bottom left) and scanning electron microscopy (top right). In particle analysis applications, light microscopy provides the number and morphology of the particles, while SEM indicates elemental composition. Here, the targeted particle is metallic and rich in tin (Sn) and nickel (Ni).

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Serial block-face imaging by in situ ultramicrotomy in an SEM. This is an iterative process in which slices (down to 15 nm thickness) from a sample block are consecutively removed with a diamond knife and the block surface imaged. The resulting stack of images can be rendered as a 3D view of the specimen. Based on earlier work by Stephen B. Leighton in 1981, this technique was implemented as shown in this video by Winfried Denk and Heinz Horstmann in 2004. Shortly afterwards commercial solutions became available.

Focused ion beam (FIB) is shown milling a spiral pattern while the SEM records its progress, demonstrating how material can be removed by the FIB and how these two modalities can work in unison.

A focused ion beam (FIB)-SEM tomography image stack of a superconducting wire. The superconducting wires are embedded in a copper matrix. During FIB-SEM tomography, the sample is progressively sectioned by the FIB, while the SEM records the images. This results in a stack of images with volumetric information.

Sample


Because SEM images with an electron beam, the sample must be either electrically conductive or some sort of charge neutralization mechanism must be provided. For some samples, such as metals and semiconductors that isn't a problem, but many non-conductive samples ("insulators") need to be sputter-coated with a fine metal coating such as gold and palladium to make them conductive.

Preparation of biological samples can be particularly challenging since, in addition to being insulating, these samples are often not compatible with the high vacuum levels in the SEM chamber. Wet samples, such as cells, are therefore fixed with glutaraldehyde and osmium tetroxide, then dried using ethanol and liquid carbon dioxide. Finally, the samples are sputter-coated and imaged.

An alternative approach is environmental scanning electron microscopy (ESEM). ESEMs enable the direct imaging of wet biological materials by incorporating design elements to bridge the substantial pressure divide between the electron optics (at high vacuum) and the sample chamber (low vacuum).

Electron source


All microscopes require a light source. In a light microscope that might be a laser or halogen lamp; in electron microscopes, it's an electron source, of which three types are common (see table below).

Thermionic emitters use a heated tungsten filament with a sharpened tip as an electron source, or alternatively, a brighter and longer-lasting lanthanum hexaboride (LaB6) crystal. The operating temperature used is higher than the onset electron emission temperature of the tip material.

Cold field-emission sources operate on a different principle: A thin, sharpened tungsten wire is positioned near a positively charged extraction electrode. The electrical potential difference between the two creates a very high electric field at the apex of the tungsten wire and causes electron emission via quantum tunneling. In contrast to cold field-emitters, in a thermally assisted or Schottky-type field-emission source, the emitting filament is heated, yielding a more stable electron beam.

Though all these designs are functional, thermionic emitters generally have a shorter lifetime and lower resolution than field emitters. But field emitters require lower (and thus more expensive and delicate) vacuum pressures, plus higher acquisition and maintenance costs.

Type Thermionic Cold field Schottky
Material W LaB6 W ZrO/W
Operating
temperature (K)		 2800 1900 Room Temperature 1800
Vacuum (Pa) ≤10-2 ≤10-3 ≤10-7 ≤10-5
Brightness
(A/cm2 sr kV)		 104 105 2 x 107 107
Maximum probe current (nA) 1000 1000 <20 100-500
Best achievable resolution (nm) < 3 < 2 < 1 < 1

Typical characteristics of different electron source types. W, tungsten; LaB6, lanthanum hexaboride; ZrO/W, zirconiated tungsten; K, kelvin; Pa, pascals; A/cm2, current density (amperes per square centimeter); sr, square radian; kV, kilovolt; nA, nanoamperes; nm, nanometers.

By: Dr. rer.nat.Alexander von Ardenne [GFDL (http://www.gnu.org/copyleft/fdl.html) or CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

© Bettmann/CORBIS

Archiv der Max-Planck-Gesellschaft, Berlin-Dahlem

Jeff Lichtman, Harvard University

SE (Everhart-Thornley) image of the arms of octopus Eledone larva.

BSE image of Ag particles on antimicrobial dressing.

CL image of Zircon.

EDS map image of a paint sample from original artwork.

STEM image of mouse brain.

EBSD pattern of silicon.