Scanning electron microscope

A scanning electron microscope (SEM) is an advanced type of electron microscope that forms highly detailed images by scanning a specimen’s surface with a focused beam of electrons. As the electrons interact with atoms near the surface, they produce signals that reveal the specimen’s topography, composition, and microstructural features. SEM technology is widely used across materials science, biology, nanotechnology, semiconductor engineering, and forensic analysis because of its exceptionally high resolution and characteristic three-dimensional imaging capability.

Historical Background

The conceptual foundations of scanning electron microscopy were developed during the early twentieth century. Max Knoll in 1935 produced the first electron beam–scanned image exhibiting channelled contrast. Shortly afterwards, in 1937, Manfred von Ardenne invented a scanning electron microscope capable of high optical resolution using a finely focused and demagnified electron beam. His work also addressed chromatic aberration in transmission electron microscopes and explored multiple detection modes and theoretical principles of SEM operation. Around the same time, Cecil E. Hall, working under E. F. Burton at the University of Toronto, constructed one of the earliest emission microscopes in North America. These early contributions eventually led to the development of modern SEM instruments capable of resolutions finer than one nanometre.

Principles and Signal Generation

SEM imaging relies on the interactions between a high-energy primary electron beam and the atoms of the specimen. As the beam rasters across the surface, several types of signals are produced from differing interaction depths:

• Secondary electrons (SE): Low-energy electrons emitted from the top few nanometres of the surface. Because of their shallow escape depth, SEs provide high-resolution images of surface topography. The Everhart–Thornley detector is the most common SE detector.
• Backscattered electrons (BSE): Beam electrons elastically scattered back from deeper within the specimen. These higher-energy electrons convey compositional contrast, as their intensity strongly correlates with atomic number. BSE imaging is valuable for distinguishing regions with differing elemental composition.
• Characteristic X-rays: Emitted when inner-shell electrons are displaced and higher-energy electrons fill the vacancies. Energy-dispersive X-ray spectroscopy (EDS) or wavelength-dispersive spectroscopy (WDS) detect these X-rays to identify and quantify elements present in the sample.
• Other signals: Cathodoluminescence, absorbed current, and transmitted electrons may also be used depending on detector configuration.

The small diameter of the electron beam affords SEM images their large depth of field, producing a distinctive three-dimensional appearance that is particularly useful for microstructural and morphological analyses.

Sample Preparation

Specimens must be compatible with the microscope’s vacuum and electron beam conditions. Preparation depends on sample composition, conductivity, and fragility.

• Mounting: Samples are affixed to a specimen stub, usually with conductive adhesive, to ensure good electrical grounding.
• Conductive coating: Non-conductive materials accumulate charge under the electron beam, causing distortions and artefacts. To mitigate this, samples are often coated with thin layers of conductive metals such as gold, gold–palladium, platinum, tungsten, iridium, osmium, chromium, or graphite. Heavy-metal coatings can also enhance secondary electron yield.
• Bulk conductivity enhancement: Biological samples may undergo osmium impregnation using OTO (osmium tetroxide–thiocarbohydrazide–osmium) techniques to increase conductivity.
• Alternative imaging conditions: Environmental SEM (ESEM) allows imaging of non-conductive or hydrated samples by maintaining a higher chamber pressure, which neutralises charge. Low-voltage SEM, often using field emission guns, reduces charging by operating at low accelerating voltages.

Embedding specimens in resin and polishing to a mirror finish is common when backscattered electron imaging or quantitative X-ray microanalysis is required.

Biological Sample Preparation

Biological specimens require more elaborate preparation because SEM chambers operate under high vacuum and living tissues collapse when dried improperly.
Typical preparation steps include:

1. Fixation: Chemical fixatives such as glutaraldehyde, formaldehyde, or combinations thereof stabilise cellular structures. Post-fixation with osmium tetroxide enhances membrane contrast.
2. Dehydration: Gradual solvent exchange, usually with ethanol or acetone, replaces intracellular water.
3. Drying: Critical point drying with liquid carbon dioxide avoids surface tension effects that would otherwise cause collapse during air-drying. Alternatively, cryogenic methods preserve delicate tissues by rapid freezing.
4. Coating: After drying, specimens are coated with conductive metals unless imaged in an ESEM.

Rigid biological materials such as wood, bone, dried insects, feathers, and shells typically require minimal preparation beyond cleaning and mounting.

Imaging Modes and Operational Conditions

SEM operation varies according to specimen requirements and the type of analysis desired:

• High-vacuum SEM: Standard mode producing high-resolution images for stable, conductive samples.
• Low-vacuum and environmental SEM: Suitable for non-conductive, wet, or volatile samples without the need for conductive coating.
• Cryogenic SEM: Enables imaging at very low temperatures to preserve hydrated or temperature-sensitive materials.
• Variable accelerating voltages: Adjustments allow optimisation of penetration depth, signal type, and surface sensitivity.

SEM instruments may also include sample stages capable of tilting, rotating, or heating, enabling complex analyses such as three-dimensional reconstruction, in situ materials testing, and semiconductor wafer inspection.

Applications and Significance

The SEM is one of the most versatile tools in modern microscopy. Its applications include:

• Surface morphology in materials research.
• Failure analysis and defect inspection in semiconductor manufacturing.
• Biological morphology and taxonomy.
• Forensic investigations.
• Geological and mineralogical characterisation.
• Industrial quality control.
• Nanotechnology and thin-film studies.