Technical Terms & Techniques

This glossary provides definitions for electron microscopy techniques, terms and acronyms – as well as links to further information on our site. The terms are listed alphabetically, and are quick-linked to their definitions so you can easily find the information you are looking for.

  • Ångström
  • Argon gas
  • Carbon Coating
  • Carbon fibre
  • Carbon Rod
  • Chromium sputter coating
  • Critical point dryer (CPD)
  • Critical point drying
  • Cryo fixation
  • Cryo transfer
  • Cryo-DualBeam
  • Cryo-electron microscopy
  • Cryo-FIB/SEM
  • Cryo-preservation
  • Cryo-scanning electron microscopy (cryo-SEM)
  • Dehydration
  • Embedding
  • Environmental scanning electron microscope (ESEM)
  • Film thickness monitor (FTM)
  • Fixation (chemical fixation)
  • Freeze drying
  • Freeze-fracture-etch
  • Glow discharge
  • Gold coater
  • High vacuum
  • Iridium sputter coating
  • Low vacuum ( Rough vacuum)
  • Low-angle shadowing
  • Magnetron sputtering
  • Micro-electromechanical systems (MEMs)
  • Micrometer ( m)
  • Nanometer (nm)
  • Pirani vacuum gauge
  • Plasma asher
  • Plasma ashing
  • Plasma etcher
  • Plasma etching
  • Rotary pump
  • Scanning electron microscope (SEM)
  • SEM cold stage (peltier-cooled)
  • Sputter coater
  • Sputter coating
  • Sputter target
  • Sublimation
  • Supercritical drying
  • TEM sectioning
  • TEM staining
  • Transmission electron microscopy (TEM)
  • Turbomolecular pump
  • Ultramicrotome
  • Vacuum evaporation
  • Vacuum evaporator
Ångström
Argon gas

Argon (Ar) is widely used as the process gas during sputter coating. The use of argon is essential for clean, contamination-free coating of scanning electron microscopy (SEM) specimens.

Carbon Coating

The thermal evaporation of carbon (C) is widely used for preparing specimens for electron microscopy (EM). A carbon source – either in the form of woven fibre or graphite rod – is mounted in a vacuum system between two high-current electrical terminals. When the carbon source is heated to its evaporation temperature, a fine stream of carbon is deposited onto specimens.

The main applications of carbon coating in EM are making scanning electron microscopy (SEM) specimens conductive for subsequent examination by X-ray microanalysis, and being used as specimen support films on transmission electron microscopy (TEM) grids.

Carbon fibre

Carbon fibre, normally in the form of a woven cord, can be used to thermally evaporate thin layers of carbon onto a substrate. The main application in electron microscopy (EM) is the production of thin, electrically-conducting coatings on scanning electron microscopy (SEM) specimens. Carbon fibre can be used for transmission electron microscopy (TEM) applications, but carbon rod is normally preferred due to superior control of the evaporation process.  For further details of please see: carbon fibre.

Carbon Rod

Carbon (C) in the form of shaped graphite rods can be used to thermally evaporate thin layers of carbon onto a substrate. Common applications in electron microscopy (EM) include the production of carbon-coated transmission electron microscopy (TEM) grid support films and the making of TEM surface replicas. Carbon rod evaporation is also used to produce electrically-conducting surface coatings on scanning electron microscopy (SEM) specimens.

For further details of please see: carbon rod.

Chromium sputter coating

Sputter coating with chromium (Cr) is widely used for depositing fine grain, high-resolution films onto field emission scanning electron microscopy (FE-SEM) specimens. Chromium oxidises on contact with air, which can present specimen storage problems. For this reason, iridium (Ir) sputter coating is increasingly preferred by many workers.

Critical point dryer (CPD)

This is an instrument used for critical point drying. For scanning electron microscopy (SEM) specimen preparation, small self-contained bench-top systems are normally used. A critical point dryer is essentially a temperature-controlled pressure vessel. The process chamber has facilities for introducing liquid carbon dioxide, and draining and venting liquids and gases. Specimens are held in a holder ( boat ) during transfer and processing.

Quorum Technologies introduced the first commercial critical point dryer (the E3000) for SEM specimen preparation in 1971, and now produce a range of these instruments.

Critical point drying

This is a process used to remove liquid from scanning electron microscopy (SEM) specimens in a precise and controlled way. Air drying would result in unacceptably high levels of structural damage, caused by the drying boundary as it passes through specimens. Critical point drying avoids these surface tension effects because the specimens pass from liquid phase to gas phase without crossing the liquid-gas boundary.

Critical point drying is primarily used to dry biological specimens prior to SEM examination, but is also increasingly used to dry micro-electromechanical systems (MEMs), which tend to be broken apart or distorted by strong surface tension forces in the drying front.

Cryo fixation

Cryo fixation is the rapid freezing of a water-based specimen to prevent or reduce growth of ice crystals within that specimen. This preserves the specimen in a snapshot of its solution state with the minimal of artefacts. An entire field called cryo-electron microscopy has branched from this technique. With the development of cryo-electron microscopy, it is now possible to observe virtually any biological specimen close to its native state.

Cryo-DualBeam
Cryo-FIB/SEM

An FIB/SEM is a combined focused ion beam (FIB) microscope and scanning electron microscope (SEM). This instrument, widely known as a DualBeamtm microscope, uses the milling action of a gallium ion beam to reveal internal surfaces and an electron beam to record successive image slices and give 3D morphology. Either secondary electrons (for maximum surface detail) or back-scattered electrons (which have atomic number contrast) can be used to form images.

The addition of a suitable cryo preparation system, such as the PP3010T, allows the observation of biological material, beam-sensitive specimens and many other wet materials, such as foods, polymers, oils, foams and greases.

Cryo-preservation
Cryo-scanning electron microscopy (cryo-SEM)

Cryo-SEM is a method for preparing liquid, semi-liquid or beam-sensitive specimens for examination in a scanning electron microscope (SEM). The technique has huge benefits compared to alternative methods, such as critical point drying and freeze drying, because all of the specimen s water is retained.

Dehydration

This is the process of removing water from electron microscopy (EM) specimens. The water is generally replaced with organic solvents, such as ethanol or acetone. This happens in an intermediate step between chemical fixation and total drying for scanning electron microscopy (SEM) specimens using the critical point drying method, or between infiltration with resin and subsequent resin embedding for transmission electron microscopy (TEM) specimens.

Embedding

This is the infiltration of the biological tissue and similar material with wax (for light microscopy) or resin (for electron microscopy). Common embedding resins include Araldite and LR White, which can then be polymerised (by heat or UV light) into a hardened block for subsequent sectioning in an ultramicrotome and observation in a transmission electron microscope (TEM).

Environmental scanning electron microscope (ESEM)

A type of scanning electron microscope (SEM) with a specimen chamber that can be adjusted to different environmental conditions, typically to observe ‘fresh’ (unprocessed) material in a vacuum or to keep specimens ‘wet’ in order to study their equilibrium with water.

Film thickness monitor (FTM)

A film thickness monitor can be used to monitor and control the thickness of sputtered and evaporated metal films. A gold-coated quartz crystal is mounted in the vacuum chamber of the coating system, ideally close to the specimen or substrate. The quartz crystal is made to oscillate at a defined frequency, using an externally-mounted oscillator. As metal is deposited on the quartz crystal, the frequency of oscillation alters and the change is converted to a digital (eg LED) display on the monitoring unit.

Film thickness monitors are available for use with most of our coating systems and cryo preparation systems.

Fixation (chemical fixation)

A general term for the process of chemically preserving specimens at a moment in time. Fixation prevents further deterioration, so that the specimen appears as close as possible to what it would be like in its original living state.

In chemical fixation for electron microscopy (EM), glutaraldehyde is widely used to crosslink protein molecules within biological specimens, and osmium tetroxide to preserve lipid content.

NB: Cryo-preservation is increasingly being used to overcome the many artefacts associated with chemical fixation.

Freeze drying

Freeze drying will reduce the distortion that occurs when a ‘wet’ scanning electron microscopy (SEM) or transmission electron microscopy (TEM) specimen dries by normal (room temperature) evaporation. Distortion is caused by large surface tension forces present when passing from a liquid to a gas phase. In the case of biological specimens, this means passing from liquid water to water vapour. If, however, the specimen is frozen and maintained in a frozen state under vacuum, frozen water can be removed by careful sublimation. This avoids the gas stage and thereby reduces specimen distortion.

Our product range includes both peltier-cooled and liquid nitrogen-cooled bench-top freeze dryers, specifically designed for electron microscopy (EM) specimen preparation. Alternative preparation techniques for SEM include critical point drying and cryo-SEM. Cryo preparation is the optimal technique as it allows observation with the specimen water in situ.

Freeze-fracture-etch

Freeze-fracture-etch generally refers to a transmission electron microscopy (TEM) preparation method requiring specialist instrumentation. It can also form part of the preparation protocol for cryo-SEM, although for surface observation fracturing it is not required.

TEM freeze-fracture preparation is useful for observing specimens such as lipid membranes and their associated proteins. The fresh tissue or a cell suspension is rapidly frozen and then fractured while maintained at a low temperature. The fractured surface is generally ‘etched’ (sublimated) by increasing the temperature to about -95 C for a few minutes to allow some surface ice sublime to reveal microscopic details. For scanning electron microscopy (SEM), the specimen is now ready for imaging.

For TEM, the specimen can then be shadowed with evaporated platinum (Pt) at low angle (about 6 ). Carbon (C) is then evaporated perpendicular to the surface plane to improve stability of the replica coating. The specimen is allowed to return to room temperature and pressure, and then the ‘shadowed’ metal replica of the fracture surface is removed from the underlying biological material by careful chemical digestion, normally using a bleach solution. The floating replica is thoroughly washed, carefully picked up on a TEM grid and viewed in a TEM.

Due to the improvements in the performance and resolution of SEMs and associated cryo-preparation systems, cryo-SEM has taken over many of the applications that were previously only possible with TEM freeze-fracture replication techniques.

Glow discharge

Electric glow discharge is a type of plasma formed by passing a current at 100 V to several kV through a gas at low pressure (i.e. in a vacuum system). The main application of glow discharge in electron microscopy (EM) is to convert naturally hydrophobic (‘water-hating’) carbon-coated transmission electron microscopy (TEM) support grids into a hydrophilic (‘water-loving’) condition. Glow discharge treatment with air will make film surfaces negatively charged and hydrophilic and allow the easy spread of aqueous solutions.

Other treatments include:

Hydrophilic-positive treatment in air with magnesium acetate post-treatment to allow nucleic acid adhesion to carbon films.

Hydrophobic-positive treatment with alkylamine for proteins, antibodies and nucleic acids.

Hydrophobic-negative treatment in air for positively charged protein molecules, (e.g. ferritin and cytochrome c).

Glow discharge can also be used for modifying surface, for example, to increase bond strength of polymers.

For further information please see the GloQube – a dedicated glow discharge system.

Glow discharge is also available on other coating systems (e.g. the SC7620Q150RQ150T and Q300 series with optional attachment) or add-ons to larger vacuum evaporators (e.g. the K975X).

NB: Glow discharges are sometimes considered to be ‘imperfect’ plasmas and cannot be used to plasma etch or plasma ash specimens their use mainly being confined to altering surface energies, not the removal of bulk material. For these applications the K1050X RF Plasma Barrel Reactor is recommended.

Gold coater

A common name for a sputter coater used for coating scanning electron microscopy (SEM) specimens with thin layers of gold (Au). Historically, gold was the most common metal used for SEM coating applications. However, as the stability and resolving power of SEMs has increased over the years, a wider range of sputter target materials with smaller grain sizes have been used. These include platinum (Pt), chromium (Cr) and iridium (Ir). An alloy of gold/palladium (Au/Pd), often 80:20, is commonly used instead of gold as it gives a film with a smaller grain size. Quorum offer a range of gold coaters to meet SEM and thin film applications.

High vacuum
Iridium sputter coating
Low vacuum ( Rough vacuum)
Low-angle shadowing
Magnetron sputtering
Micro-electromechanical systems (MEMs)
Micrometer ( m)
Nanometer (nm)
Pirani vacuum gauge
Plasma asher
Plasma ashing
Plasma etcher
Plasma etching
Rotary pump
Scanning electron microscope (SEM)
SEM cold stage (peltier-cooled)
Sputter coater
Sputter coating
Sputter target
Sublimation
Supercritical drying
TEM sectioning
TEM staining
Transmission electron microscopy (TEM)
Turbomolecular pump
Ultramicrotome
Vacuum evaporation
Vacuum evaporator


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