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1. ion etching

"Ion etching" is a technique to selectively remove specific atoms and the atoms at lattice defects. This technique is used for observation of textures on a specimen surface. A technique of uniform and nonselective thinning for specimen preparation of TEM is ion milling. Sometimes, ion etching is confused with ion milling.

Related term
ion milling

2. ion cleaner

An ion cleaning tool that removes contaminants on a TEM specimen, which are residual substances deposited on a specimen at specimen pre-treatment, and contaminated substances deposited on a specimen in specimen storage. The specimen holder on which a specimen is set is inserted into an "ion cleaner." Then, a glow discharge due to residual gasses in the ion cleaner is generated and the ionized gasses by discharge irradiate the specimen. This chemical-reaction of the ionized gasses removes the contaminants (in particular, polymerized substances of hydrocarbons) deposited on the specimen.

Related term
contamination, plasma cleaning

3. ion sputtering

"Ion sputtering" is a phenomenon where atoms are sputtered from a solid surface when ionized and accelerated atoms or molecules hit the solid surface. This phenomenon is utilized for formation of a thin film on a solid surface, specimen coating and ion etching. In the case of specimen coating, discharge is generated in a low vacuum or in an Ar gas environment under a vacuum, the ionized gas is accelerated and hits a target material (Au, Pt, etc.) at the anode. Then, the material is emitted from the target surface and deposit on a specimen at the cathode, specimen being coated.

Related term
focused ion-beam milling, FIB

4. milling

4-1. milling

"Milling" is a technique to mill and fabricate a specimen substance. In specimen preparation for TEM, an ion beam is used for milling.

4-2. ion milling

"Ion milling" is a specimen preparation technique that is used when electrolytic polishing or chemical polishing cannot be used to prepare a specimen. This technique is particularly effective for the specimen preparation when cross-section observations of layered materials are requested. A thin cross sectional film is prepared by milling surface layer atoms with the irradiation of an argon ion beam accelerated at 2 kV to 10 kV with a grazing incidence angle less than 10°. The disadvantage of ion milling is that damage to the specimen is unavoidable. Commercially-available ion-milling instruments are equipped with an optical microscope or a CCD camera with a magnification of several ten times to view the state of the specimen.

Related term
focused ion-beam milling, FIB, gentle milling, electrolytic polishing, chemical polishing

4-3. focused ion-beam milling

"Focused Ion Beam (FIB) milling" is a technique of a TEM specimen preparation to mill a bulk specimen with focused gallium (Ga) ions. The target region of the bulk specimen can be selectively thinned down to a desired shape while monitoring and controlling by SEM observation of the milling region. This technique is particularly indispensible for failure analysis of semiconductor devices. The FIB milling procedure is as follows: First, the surface of the target region is coated with a platinum- or carbon-protective film in the FIB system to avoid the milling of the target region (Fig. (a)). Next, the target region is milled with a Ga ion beam of a high accelerating voltage of about 30 kV to prepare a section of a thickness of a few μm or less (Fig. (b)). Then, the prepared section is picked up from the bulk specimen and fixed onto a TEM specimen grid (Fig. (c)). Finally, the fixed specimen is milled with a Ga ion beam of a low accelerating voltage of about 5 kV (for decreasing damages to the specimen) to create a thin section of a thickness of 10 to 100 nm (Fig. (d)). If the damaged layers due to the irradiation of Ga ions remain, the layers are removed by another milling with a Ga ion beam of a lower accelerating voltage (about 3 kV or less) or with an argon (Ar) ion beam. Focused ion-beam milling.png

Fig. (a) Protection layer is formed on the target region by coating of carbon or platinum. (b) Surroundings of the target region are roughly milled with Ga ion beam to prepare a section (thickness: a few μm or less). (c) A section is fixed onto a TEM grid. (d) Thin section (thickness: 10 to 100 nm) is created from the target region by fine milling. (e) The thinned section prepared by FIB is subjected to TEM observation.

Related term
ion milling, gentle milling

5. plating

5-1. plating

"Plating" is a technique to form thin metal layers deposited on a surface of a solid.

Related term
ion plating, electrolytic plating

5-2. ion plating

"Ion plating" is a technique to form a thin metal layer deposited on a surface of a solid by ionizing metal. In this technique, discharge is generated in a low vacuum or a gas environment between the substrate acting as a cathode and a metal evaporation source acting as an anode. Then, metal atoms are evaporated and ionized, and accelerated by an electric field, then finally form a thin layer on the substrate.

5-3. electrolytic plating

"Electrolytic plating" is a technique to form a thin metal layer on a surface of a solid specimen by electrolysis of a conductive solid (ex. metal) in a suitable electrolyte solvent. Actually, a specimen (acting as an anode) and a platinum (Pt) plate or a stainless-steel (acting as a cathode) are immersed in a suitable electrolyte solvent. By the flow of electric current between these electrodes, the eluted Pt or stainless-steel is deposited on the specimen surface.

6. liquid-metal ion source

A "liquid-metal ion source" uses a metal that is at the liquid state at normal temperature. This ion source, normally liquid gallium, is used for an FIB instrument.

Related term
focused ion-beam milling, FIB

7. etching

7-1. etching

"Etching" is to selectively remove specific surface atoms of a specimen by utilizing chemical or physical reactions.

Related term
ion etching, polishing

7-2. freeze etching

Freeze etching is a technique to expose the structure under ice on a freeze-fractured surface of a biological specimen, by sublimating the ice under vacuum. After the structure is exposed, a replica is made from the fractured surface for TEM observation. This technique is mainly used to observe the structures of cytoskeletons, organelles, etc.

Related term
freeze fracturing, freeze replication

7-3. ion etching

"Ion etching" is a technique to selectively remove specific atoms and the atoms at lattice defects. This technique is used for observation of textures on a specimen surface. A technique of uniform and nonselective thinning for specimen preparation of TEM is ion milling. Sometimes, ion etching is confused with ion milling.

Related term 
ion milling

8. epitaxy

8-1. epitaxy

"Epitaxy" is a technique to deposit and grow a crystalline film on a crystalline substrate using vacuum evaporation (deposition) or chemical vapor deposition (CVD), etc. In epitaxy, the crystalline film formed has a specific orientation relation with the substrate crystal.

Related term
molecular beam epitaxy, MBE

8-2. molecular beam epitaxy

"Molecular beam epitaxy (MBE)" is a technique to deposit and grow a crystalline film in an ultra-high vacuum. In MBE, target elements or target materials that will constitute a target crystal are heated and evaporated and then, the target crystalline film is deposited and grown on a heated crystalline substrate. MBE is similar to vacuum evaporation (deposition), but the technique accurately controls the molecular beam to prepare a crystalline specimen controlled at the atomic scale. MBE is used also for specimen preparation for a TEM.

Related term
vacuum evaporation (deposition)

9. grid

9-1. grid

A "grid" is a plate of a metal, etc. with a diameter of 3 mm and a thickness of 20 to 50 μm, which supports a specimen for TEM observation. Various grids such as a square-, a circular-, and a slit-grid, are available. They are selectable depending on observation requirements. The material of the grid is copper (Cu), molybdenum (Mo), gold (Au) or titanium (Ti), etc. In elemental analysis, a grid which does not contain the elements to be analyzed, is used.

1.Reticular grid The grid is most commonly used. Fragmented specimens are placed or pasted on the grid. For viruses or small particles, a supporting film is pasted on the grid and then, the specimen is placed on it.

2.Single hole grid The grid is used to observe a wide area of specimen without disturbance of the mesh of a reticular grid. Thus, it is suitable for observation at ultra-low magnifications.

3.Reference grid The grid is conveniently used at repeated observation of the same field because the marks (letters) to memorize the observation position are prepared on the grid.

4.FIB grid The grid is used to attach a thin film specimen prepared by FIB, to the heads of the grid.Related term

Grid.jpg

Related term
supporting film, micro grid

9-2. microgrid

A "microgrid" is a supporting film used to support a fine specimen like powder for a TEM. The microgrid is made of cellulose aceto-butyrate and has many holes with the hole diameter of several μm or less.

Related term
grid, supporting film

10. polishing

10-1. polishing

"Polishing" is a mechanical or chemical treatment to prepare a smooth and uniform surface without selecting crystal orientations unlike etching and cleaving.

Related term
mechanical polishing, chemical polishing, electrolytic polishing, etching

10-2. electrolytic polishing

"Electrolytic polishing" is a technique used for preparing a thin film on metal, alloy, etc. In this technique, a specimen (acting as an anode) and a platinum (Pt) plate or a stainless-steel (acting as a cathode) are immersed in a suitable electrolyte solvent. Then, an electrostatic potential is applied between these electrodes to elute the atoms of the specimen surface, and finally the specimen surface is thinned with its surface kept smooth. The advantage of electrolytic polishing is that a thinned specimen can be prepared with no mechanical stress.

Related term
jet polishing, chemical polishing, ion milling

10-3. jet polishing

"Jet polishing" is one technique in electrolytic polishing. In this technique, a jet-like polishing solution is sprayed onto a specimen to make a hole at its center. Spraying the solution makes it possible to keep the solution at the specimen surface fresh, thus a quick and smooth polishing is achieved.

Related term 
electrolytic polishing, chemical polishing, ion milling

10-4. chemical polishing

"Chemical polishing" is a technique used for thinning inorganic materials of semiconductors and insulators. In this technique, a specimen is immersed in a polishing solution that is mainly composed of strong acid or strong alkali. Then, the specimen is thinned with its surface kept smooth. The advantage of chemical polishing is that thin specimens can be made with no mechanical distortion stress. Normally, a polishing solution that does not cause selective dissolution (etching) is used. When a particular layer in a multi-layer semiconductor is required to observe, a specified solution is used, which dissolves other layers while leaving the particular layer.

Related term
electrolytic polishing, ion milling 

10-5. mechanical polishing

"Mechanical polishing" is a physical polishing technique used for specimen preparation of TEM. The common techniques are as follows: (1) Manual polishing using water-resistant paper (down to ~100 μm thick), (2) Rotational polishing device-based polishing using diamond or corundum particles (down to ~several 10 μm thick), (3) Dimple grinder-based polishing using corundum particles (down to <10 μm thick), and (4) Tripot polisher-based polishing using diamond particles (down to <10 μm thick).

elated term
tripot polisher, dimple grinder

11. coating

11-1.coating

"Coating" is to form a thin layer (ex. metallic layer) on the surface of a target substance.

Related term 
conductive coating

11-2.conductive coating

"Conductive coating" is to coat a nonconductive specimen for TEM with conductive carbon by arc discharge so that charging on this specimen is prevented and observation is properly performed.

Related term
charging

12. ice embedding

Ice embedding is one of rapid freeze fixation techniques used for biological specimen preparation in transmission electron microscopy. This technique embeds a suspended specimen of biological macromolecules (purified proteins, viruses, etc.) in an amorphous ice film with a few tens of nm to a few hundreds of nm. It enables us to observe a molecular state of the specimen in a liquid solution without staining. In an actual experiment, a few μL suspension liquid is dropped onto a micro grid subject to hydrophilic treatment, excessive liquid is removed with a filter paper, then the specimen is rapidly frozen by immersing it into a coolant as quickly as possible. Liquid ethane or liquid propane is used commonly as a coolant because these are easy to sublimate and have a large temperature difference between the melting point and the boiling point.

Schematic of ice embedding.

  • Ice embedding a.jpg

Cross sectional view of ice-embedded specimens, which are embedded in amorphous ice.

  • Ice embedding b.jpg

Cryo-TEM image of ice-embedded bacteriophage T4.

  • Ice embedding c.jpg

The morphology of the head and tail parts of the bacteriophage is well preserved.

Related term
cryo-electron microscopy, rapid freeze fixation, amorphous ice, high pressure freezing, metal mirror freezing (slam freezing)

13. top surface

In a solid surface, the "top surface" is defined as an extremely thin surface (~0.5 to several nm from the surface). This term is used in the following manner. (1) The top surface of a specimen is fabricated. (2) Auger electrons are emitted from the top surface.

14. gentle milling

A surface of a specimen milled by the focused ion beam (FIB) technique or a normal ion milling technique is likely to suffer damage. To remove the damaged layers on the specimen surface, an argon ion beam at a low accelerating voltage of 100 V to 2 kV is used to gently mill the specimen surface. This technique is termed "gentle milling."

Related term
focused ion-beam milling, FIB, ion milling

15. supporting film

A "supporting film" is a thin carbon, polyvinyl formal, or collodion film with a thickness of several 10 nm or less. It is pasted on a grid to support a specimen for a TEM on the specimen holder.

Related term
grid, microgrid

16. focused ion-beam milling

"Focused ion beam (FIB) milling" is a specimen preparation technique for TEM, which is used to mill a specific area on a specimen, such as a defective part on a semiconductor device. The specific area is fabricated to a wedge shape with a high accuracy of sub-micrometer scale. A focused gallium ion beam accelerated at several kV to 40 kV irradiates a specimen to mill the specific area while this area is monitored through a secondary ion image (a SEM image can also be viewed with some FIB instruments), resulting in formation of a thin section specimen. Since damage to the specimen caused by gallium ions is unavoidable, ion milling at a low accelerating voltage (gentle milling) may be applied afterwards to remove damaged layers on the specimen surface.

Related term
ion milling, gentle milling

17. sintering

"Sintering" is to heat a powder specimen until each powder in the specimen adheres to each other without melting.

18. filler

A material used for sintering. Yttria, etc., are used as "filler.".

19. radiation damage

"Radiation damage" is defined as structure deterioration of a specimen due to the irradiation of an electron beam. The primary radiation damage includes the knock-on damage and the ionization damage. In the knock-on damage process, an atom that suffers the collision by an incident electron is ejected from its lattice site, forming an interstitial atom and an atomic vacancy at the corresponding lattice site. In the ionization damage process, outer-shell electrons of an atom are ejected and the atom is ionized. In a subsequent relaxation process after the primary radiation damage process, particular lattice defects or amorphous structures are created. This process is called the secondary radiation damage process.

20. contamination

20-1. contamination

Hydrocarbons deposited on a specimen surface condense at an electron-beam irradiation area by the electrostatic force of the electron beam and are polymerized by the electron beam. This phenomenon is called "(specimen) contamination." Contamination is particularly severe when a small specimen area is irradiated with a focused, high-density electron beam, for example CBED or STEM.

Related term
hydrocarbon

20-2. anti-contamination device

A device that is used for suppressing the deposition of hydrocarbons on a specimen in a TEM. By the condensation action of the cooled fin with liquid nitrogen which is set to surround the specimen holder, contamination on the specimen due to hydrocarbons is largely decreased. In this device, two small aperture holes are placed at the top and bottom to allow the electron beam to pass through the holes. The tip of the specimen holder (specimen part) is inserted into the fin through the opening at its lateral side.

Related term
contamination

21. specimen environment

An environment when a specimen is observed or analyzed in TEM. Various specimen environments are used, which include a high-temperature environment, a low-temperature environment, an atmospheric pressure environment, and an environment with tensile force, electric fields or magnetic fields.

22. specimen preparation

"Specimen preparation" is to prepare a suitable specimen in observation and analysis of TEM.

23. deposition

23-1.deposition

"Deposition" is to form a thin film on a solid surface by physical sputtering, chemical vapor deposition, etc.

Related term
ion sputtering, ion plating, vacuum evaporation (deposition), molecular beam epitaxy, MBE

23-2. vacuum evaporation (deposition)

"Vacuum evaporation (deposition)" is a technique to form a thin substance layer on a surface of a substrate by evaporating the substance in high vacuum. For a TEM, this technique is used for specimen preparation of a metal and an alloy with uniform thickness.

Related term 
vacuum evaporator

24. vacuum evaporator

A "vacuum evaporator" is an instrument to form a thin substance layer on a surface of a substrate by vacuum evaporation (deposition). The vacuum evaporator consists of a heating element, a case and a substrate in a vacuum chamber. A substance (in most cases, a metal) that is placed in the case is evaporated by melting at high temperature, and a thin film (layer) is formed on the substrate. In specimen preparation for a TEM, a heating element, such as a tungsten (W) wire or a tantalum (Ta) plate, is resistively-heated and the substance is evaporated (deposited).

Related term
vacuum evaporation (deposition)

25. charging

If a specimen has no conductivity, part of incident electrons accumulate on the specimen. This phenomenon is called "charging." Charging disturbs normal transmission and scattering of the incident electrons, thus abnormal contrast and distortion of the image occur. To prevent charging, conductive coating is performed.

Related term 
conductive coating

26. diamond knife

A diamond knife is used to prepare an ultrathin section in ultramicrotomy. The diamond knife was originally developed to cut hard inorganic materials, which cannot be cut with a glass knife. Today, the diamond knife is used also for resin specimens because the glass knife is damaged even by a resin specimen, that is, the blade edge of the knife is likely to become dull. Thus, the diamond knife currently plays an essential role in preparing a uniform ultrathin section.

Related term
ultramicrotomy, glass knife, ultrasonic knife, ultrathin section

27. ultrasonic cleaning

"Ultrasonic cleaning" is a technique to remove contaminants on a specimen and parts of a TEM. In "ultrasonic cleaning," a cleaning solution is vibrated by an ultrasonic transducer and hits the object to clean. Since the cleaning solution can enter the fine gaps of the object, ultrasonic cleaning is suitable for precision components. Acetone is used as a cleaning solution in many cases.

28. dimple grinder

"Dimple grinder" is a mechanical polishing device. It creates a dimple (dip) with a thickness down to 10 μm or less on a parallel plate specimen prepared by polishing using a rotational polishing device (thickness: 100 μm or less). It uses a polishing agent, such as corundum particles having different particle sizes (0.1 μm to several μm). The specimen with the dimple is further thinned by ion milling.

Related term
tripot polisher, ion milling

29. electrolytic plating

"Electrolytic plating" is a technique to form a thin metal layer on a surface of a solid specimen by electrolysis of a conductive solid (ex. metal) in a suitable electrolyte solvent. Actually, a specimen (acting as an anode) and a platinum (Pt) plate or a stainless-steel (acting as a cathode) are immersed in a suitable electrolyte solvent. By the flow of electric current between these electrodes, the eluted Pt or stainless-steel is deposited on the specimen surface.

30. freeze sectioning

Freeze sectioning is used to prepare a sectioned biological specimen of soft textures. The specimen is frozen under liquid-nitrogen temperature to cut the specimen easier with a cryo-microtome. A cryo-electron microscope is needed for specimen observation.

Related term 
cryo-electron microscopy

31. conductive coating

"Conductive coating" is to coat a nonconductive specimen for TEM with conductive carbon by arc discharge so that charging on this specimen is prevented and observation is properly performed.

Related term
charging

32. tripot polisher

"Tripot polisher" is a mechanical polishing device that consists of three legs with micrometers and a leg for supporting a specimen. First, a bulk specimen is set on the leg for the specimen, and the polishing angle is finely adjusted by the micrometers, the tripot polisher is mounted on a rotational polishing device, and then the specimen is polished while the tripot polisher is supported with human fingers. A thinned specimen with a thickness of ~10 μm can be obtained from a bulk specimen (~3 mm × ~3 mm × ~2 mm thick). As a polishing material, lapping film coated with diamond of 0.1 μm to 30 μm is used. In the finishing process, the polished specimen with a mirror surface is fabricated using colloidal silica. For TEM observation, the specimen is usually polished to wedge-shaped. Normally, the specimen is finished by ion milling. Since the use of the tripot polisher enables preparation of a wedge-shaped specimen over a wide area, the device is particularly suitable for cross-sectional specimen preparation.

Related term
dimple grinder, ion milling

33. thinning

"Thinning" is to fabricate a specimen to a thin film for a TEM.

34. crushing

"Crushing" is a technique used for preparing a thin film on an oxide, a ceramic material, etc. In this technique, a bulk specimen is crushed in an agate mortor and the crushed specimen is dispersed in an organic solvent (methanol, acetone, etc.). After this process, an obtained suspending solution is dropped onto a microgrid for TEM and then thin specimen fragments are deposited.

Related term 
electrolytic polishing, chemical polishing, ion milling

35. molecular beam epitaxy

"Molecular beam epitaxy (MBE)" is a technique to deposit and grow a crystalline film in an ultra-high vacuum. In MBE, target elements or target materials that will constitute a target crystal are heated and evaporated and then, the target crystalline film is deposited and grown on a heated crystalline substrate. MBE is similar to vacuum evaporation (deposition), but the technique accurately controls the molecular beam to prepare a crystalline specimen controlled at the atomic scale. MBE is used also for specimen preparation for a TEM.

Related term
vacuum evaporation (deposition)

36. cleaving

"Cleaving" is a technique used for specimen preparation for a TEM, which cleaves a solid to expose a particular orientational section of a crystal.

37. ultramicrotomy

A specimen preparation method that is used for thinning biological specimens, high-polymer specimens and composite materials. “Ultramicrotomy” is also used to prepare thin films of soft metals. The use of a diamond knife enables a specimen to be sequentially cut with a thickness of several 10 nm to 100 nm.

Related term
diamond knife, electrolytic polishing, chemical polishing, ion milling

38. annealing

"Annealing" is a thermal treatment to heat metallic materials and others at an appropriate temperature and then to cool slowly them for obtaining uniform structures or for removing the internal stress of the materials.

39. coolant (refrigerant)

A substance that is used for cooling components of instruments or other materials. For cooling, the latent heat of the "coolant" is used, which is emitted or absorbed at the transformation from the liquid state to the gas state or vice versa.

40. replica

40-1. replica

A "replica" is a reproduction of a topographic shape of the surface of a substance (specimen). To make a replica, a thin layer of a plastic material or an inorganic material is formed on the surface of the substance, and then, this thin layer is removed, the topographic shape of the surface being copied. The replica technique is used for a reproduction of a grating or observation of the topographic shape of the specimen.

40-2. freeze replication

Freeze replication is a technique to make a metallic thin-film replica from a fractured surface of a biological specimen. When a lipid bilayer of the cell membrane is fractured, the extracellular leaflet of the freeze-fractured lipid bilayer is called the E face (or the extracellular face), whereas the cytoplasmic leaflet is called the P face (or the protoplasmic face). Thus, the technique is used to observe the morphology of membrane proteins.

Related term
freeze fracturing, freeze etching

41. rapid freeze fixation

Rapid freeze fixation is a technique to physically fix the morphology of biological tissues, biological cells, bacteria and suspended specimens composed of viruses, purified proteins, etc., by rapidly freezing the specimens. Chemical fixation, which is used in ordinary specimen preparation of biological tissues for TEM, may cause outflow of mineral salts, etc., in the tissues or deformation of the membrane structures of the tissues. On the other hand, rapid freeze fixation enables us to fix a specimen while preserving the morphology of the tissues. Rapid freeze fixation techniques include high pressure freezing, metal mirror freezing (slam freezing) and ice embedding.

Related term
high pressure freezing, metal mirror freezing (slam freezing), ice embedding, amorphous ice, chemical fixation

42. high pressure freezing

High pressure freezing, one of rapid freeze fixation techniques, is to fix biological specimens such as biological tissues, biological cells and bacteria. Rapid freezing under an about 2000 atm suppresses formation of ice crystals, which give rise to the destruction of the tissues, by decreasing the melting point of water by about -20 °C and increasing its viscosity. This technique provides a uniform freezing under amorphous ice with a depth more than one order of magnitude larger (about 200 μm) compared with the freezing depth at the atmospheric pressure. Specimen preparation for TEM observation of the frozen specimen is carried out by one of the following three procedures. (1) Applying freeze sectioning to the specimen, (2) Applying resin embedding and ultrathin sectioning to the specimen after the specimen is subject to freeze substitution and is returned to room temperature, and (3) Making a replica of the specimen by freeze fracturing.

Related term
rapid freeze fixation, amorphous ice, freeze substitution, freeze fracturing

43. amorphous ice

Amorphous ice means ice that does not take a crystalline state. When water is cooled below the melting point, ice crystal is formed and grows until its temperature reaches the re-crystallization temperature. Since formation of ice crystals can cause destruction of fine structures of a biological specimen, amorphous-ice formation without formation of ice crystals is required when rapid freeze fixation is applied. Thus, ice-crystal formation should be suppressed by cooling the specimen as rapid as possible down to the re-crystallization temperature.

Related term
rapid freeze fixation, high pressure freezing, freeze substitution, metal mirror freezing (slam freezing), cryo-electron microscopy, ice embedding

44. freeze substitution

Freeze substitution is a technique to replace amorphous ice with an organic solvent (acetone, etc.) (dehydration) in a biological specimen fixed by rapid freeze fixation, where the specimen is subject to rapid freeze fixation (high pressure freezing or metal mirror freezing (slam freezing)). To prevent destruction of fine structures of the specimen, the substitution is carried out by raising temperature step-by-step from -80 ℃ to 4 °C over a few days. In the course of the substitution, chemical fixation and electron staining are often performed by adding osmium tetroxide and/or uranium acetate. Then, the substituted specimen is returned to room temperature and is subject to resin embedding. A TEM specimen is made by ultrathin sectioning the specimen.

Related term
rapid freeze fixation, metal mirror freezing (slam freezing), high pressure freezing, amorphous ice

45. metal mirror freezing (slam freezing)

Metal mirror freezing (slam freezing) is one of rapid freeze fixation techniques. This technique punches and rapidly freezes a biological specimen against a metal block cooled by a coolant such as liquid nitrogen. In many cases, high-thermal-conductivity, high-purity copper with gold plating is used for a metal block. To increase a temperature drop efficiency of the specimen, the surface of the metal block is prepared into a flat mirror-surface. This technique can be performed using a relatively inexpensive device, but provides a smaller freezing depth suitable for TEM observation (about 20 µm) than the suitable depth produced by high pressure freezing. This technique is mainly used to fix tissues. Specimen preparation for TEM observation of the frozen specimen is carried out by one of the following three procedures. (1) Applying freeze sectioning to the specimen, (2) Applying resin embedding and ultrathin sectioning to the specimen after the specimen is subject to freeze substitution and is returned to room temperature, and (3) Making a replica of the specimen by freeze fracturing.

Related term
rapid freeze fixation, high pressure freezing, amorphous ice, freeze fracturing

46. freeze fracturing

Freeze fracturing is a technique to cut and expose the inner face of a biological specimen such as cells and tissues. This technique freezes the specimen with liquid nitrogen, etc., and then fractures the specimen under vacuum by giving an impact on the frozen specimen using a knife. After the specimen is fractured, a replica of a fractured surface is made for TEM observation. To observe the fractured surface with a TEM, either of two replication techniques is applied. One is freeze replication to make a replica of the fractured surface without any processing. Another is freeze etching by which ice on the fractured surface is sublimated, followed by making a replica of the exposed inner structure without ice. A replica is usually made under vacuum by depositing platinum or platinum palladium onto the specimen and then by depositing carbon onto the already deposited surface. The specimen is immersed and dissolved in an alkali reagent. Only the replica is mounted on a mesh (grid) for TEM observation.

Related term 
freeze replication, freeze etching

47. chemical fixation

Chemical fixation is a technique to fix a specimen with chemicals to prevent autolysis by the action of enzymes and deformation of morphologies during specimen preparation. Biological tissues start autolysis caused by their enzymes immediately after stopping the activities of them. For the TEM observation of a biological specimen, the specimen must be prepared by removing water to preserve its morphologies under vacuum in the microscope column, and by thinning the specimen to transmit an electron beam. For these requests, dehydration, resin embedding and thin sectioning are applied, but the procedures cause deformation of fine structures of the specimen. Chemical fixation is carried out to preserve the morphologies and physical properties at the living states of the specimen as much as possible. This technique prevents the autolysis and deformation by cross-linking the proteins or lipids of biological materials using chemicals. Chemical fixation for a TEM is performed by the two steps, prefixation and postfixation.

Related term 
rapid freeze fixation, prefixation, postfixation, cross-linking

48. cross-linking

“Cross-linking” means that target chain-like macromolecules are additionally bounded to each other with other molecules by constructing inter- or intra-molecular bridges.

Related term
chemical fixation, prefixation, postfixation

49. prefixation

Prefixation is the first-step fixation technique of chemical fixation for biological specimens observed with a TEM. This technique aims at inactivation and preservation of proteins. In the preparation of a biological specimen for TEM observation, dehydration and resin embedding are applied. Under these procedures, a robust fixation of the fine structures of biological tissues is required. For such a fixation, it is effective to use strong oxidizing reagents (osmium tetroxide, potassium permanganate, etc.). However, these reagents have disadvantages that include slow permeation into the specimen and destruction of proteins in the specimen. To avoid these disadvantageous phenomena, the specimen is pre-fixed with paraformaldehyde or glutaraldehyde, which permeate quickly the specimen and cross-links the proteins to preserve the original morphologies.

Related term
chemical fixation, postfixation, cross-linking

50. postfixation

Postfixation is the second-step fixation technique of chemical fixation for biological specimens observed with a TEM. This technique aims at preservation of lipids or enhancement of a TEM image contrast. In the preparation of a biological specimen for TEM observation, dehydration and resin embedding are applied. In these procedures, fixation only with aldehyde (glutaraldehyde, paraformaldehyde, etc.) cannot fix lipids in the specimen. Thus, the membrane structures that consist of lipids flow out of the specimen when performing dehydration with ethanol or an organic solvent. In addition, aldehyde bounded to the specimen does not contribute to the enhancement of the image contrast because it consists of only light elements. These problems are solved by postfixation with heavy metal oxide reagents (osmium tetroxide, etc.) which is a subsequent process of prefixation with aldehyde. Osmium tetroxide fixes lipids by binding it to a double-binding region of unsaturated fatty acid and by constructing a cross-link for preventing outflow and deformation of the lipids. In addition, osmium is a heavy metal, thus enhancing the image contrast of the binding region. Osmium tetroxide is an effective reagent, but it has disadvantages that include slow permeation into the specimen and destruction of proteins in the specimen. To prevent these disadvantageous phenomena, fixation (prefixation) of proteins with aldehyde is required before applying fixation with osmium tetroxide.

Related term
chemical fixation, prefixation, cross-linking

51. ultrasonic knife

An ultrasonic knife is a diamond knife that thins a specimen while vibrating the blade head of the knife using ultrasonic wave. In ordinary thinning with a diamond knife, a pressure can be applied to the cutting direction of the specimen. This may cause “compression” (deformation of the specimen due to compression in the up-and-down direction) or falling of granules with different hardnesses such as lipid droplets. The ultrasonic knife which vibrates its blade head right-and-left in cutting, makes it possible to disperse the pressure to the specimen. Owing to this feature, the ultrasonic knife prevents “compression” or the falling of the granules.

Related term
ultrathin section, diamond knife, ultramicrotomy

52. ultrathin section

52-1. ultrathin section

An ultrathin section means a section sliced thin enough to transmit an electron beam. In many cases, the ultrathin section has a thickness of 100 nm or less.

Related term
diamond knife, ultramicrotomy

52-2. interference color of ultrathin section

keyword “interference color of ultrathin section” Interference color of an ultrathin section is produced by the interference of light reflected from the top- and bottom-faces of the ultrathin section when white light is incident onto a section in the almost vertical direction. Since the interference color corresponds to the thickness of the section, the interference color enables us to know a rough thickness of the section. Sections with thicknesses of about 70 nm, about 100 nm and about 200 nm emit interference colors of silver gold, gold and blue, respectively.

Related term
ultrathin section, ultramicrotomy, diamond knife

53. glass knife

A glass knife is a blade for a microtome. The knife is a fabricated sharp edge line of a glass made by fracturing a thick glass or a glass rod. The glass knife is used to make an exposed specimen surface flat prior to ultrathin sectioning using a diamond knife, or to perform trimming of the resin of a resin embedded specimen. Precautions for use; When the knife is applied to hard materials or the same position of the blade is repeatedly used, the blade tends to spill and can damage the surface of a specimen. Thus, the knife needs to be used while changing the position of the blade.

Glass knife-1.jpg

Related term
diamond knife, ultrathin section, ultramicrotomy

54. electron staining

Electron staining means to adsorb heavy metals of a high scattering power to biological specimens or polymer materials composed of light elements which exhibit a small scattering power for electrons, for enhancing the TEM image contrast of these specimens. When observing a biological specimen, uranium or lead is adsorbed to proteins, etc., in the specimen. For a specimen of polymers (e.g. polypropylene, polyethylene) consisting of crystalline and amorphous states, ruthenium can be selectively adsorbed to the amorphous regions because ruthenium tetroxide enters only to the amorphous regions. Those heavy metals which strongly scatter incident electrons give rise to enhancement of the image contrast.

Related term 
negative staining, en bloc staining, scattering contrast

55. negative staining

Negative staining is one of electron staining techniques. This technique leaves heavy metals at the gaps in a specimen and on the supporting film at the surrounding regions of the specimen. Negative staining enables enhancement of the TEM image contrast.

  • Electron staining 01.jpg

Electron staining 02.jpg TEM image of negatively stained bacteriophage T4

An aqueous solution of viruses or purified proteins, etc. is dropped onto a supporting film, and then excessive water is removed from the specimen with a filter paper. Immediately after the removal of excessive water, a staining solution containing heavy metals (uranium acetate, phosphotungstic acid, etc.) is dropped onto the specimen. Then, water is removed from the specimen with a filter paper to dry the specimen, leaving the heavy materials at the gaps and on the supporting film at the surrounding regions of the specimen. As a result, these regions appear dark due to strong scattering of incident electrons, and the morphology of the specimen is elucidated. Since the specimen itself is not stained, this technique is termed "negative" staining.

Related term
electron staining, scattering contrast

56. en bloc staining

En bloc staining is one of electron staining techniques. A biological specimen (block tissue) is immersed in a solution of uranium acetate or an aqueous solution of lead aspartic acid after postfixation and before dehydration, for electron staining. En bloc staining enhances the TEM image contrast of membrane structures or fibrous structures in cells.

Related term
electron staining, scattering contrast

57. shadowing

Shadowing is a technique to obliquely deposit metals (platinum, etc.) of a high scattering power with a low angle to the specimen surface of a biological specimen composed of proteins, DNA, viruses, etc., under vacuum. By shadowing, the TEM image contrast due to roughness of the surface of those specimens is enhanced. Biological molecules, such as proteins, DNA and viruses, are composed of light elements of carbon, nitrogen, oxygen or hydrogen. When a specimen of these molecules is observed with a TEM, most incident electrons pass through the specimen without suffering scattering. As a result, an obtained image hardly shows contrast. To solve this problem, metals (platinum, etc.) are vacuum-deposited onto the molecule particles. This deposition makes it possible to enhance the contrast of the surface roughness of fine structures. As the deposition angle is lower, the finer structures can be observed. When the deposition angle is higher, the fine structures are more embedded in the deposited metal. The technique is called "shadowing" or "shadow casting" because the metal-deposited part in the TEM image appears as a shadow.

Related term
electron staining, scattering contrast

58. immunoelectron microscopy

Immunoelectron microscopy is a technique to visualize the locations (localization) of specific proteins with a TEM, by utilizing antigen-antibody reactions where antibodies bind specifically to antigenic proteins. First, antibodies (primary antibodies) react with the target proteins. Then, the secondary antibodies labeled with gold colloid, etc., react with the primary antibodies, so that the target proteins can be detected with the TEM. Immunoelectron microscopy is classified into two methods depending on the timing of antibody reactions; pre-embedding (before resin embedding) and post-embedding (after resin embedding).

Related term
pre-embedding, post-embedding

59. pre-embedding

Pre-embedding is a technique of embedding in the course of immunoelectron microscopy for the primary antibodies to react with the target antigenic proteins immediately after fixation of block tissues or cells. The name of “pre-embedding” originates from the fact that the immune-reaction is performed prior to resin embedding. The advantage of pre-embedding is that antibodies can react with the antigenic proteins in advance of the occurrence of denaturation or outflow of the antigenic proteins. However, pre-embedding has two major disadvantages. One is that antibodies do not stain uniformly the whole block tissue because staining is made for the block tissue prior to resin embedding. Another is that a large amount of expensive antibodies is needed for pre-embedding. In general, pre-embedding is a simpler technique than post-embedding.

Related term
immunoelectron microscopy, post-embedding

60. post-embedding

Post-embedding is a technique of embedding in the course of immunoelectron microscopy for the primary antibodies to react with the target antigenic proteins after ultrathin sectioning. The name of “post-embedding” originates from the fact that the immune-reaction is performed after ultrathin sectioning (subsequent to resin embedding). Post-embedding has two major advantages. One is that the locations (localization) of the target proteins are precisely elucidated because the primary antibodies react with the proteins exposed on an ultra-thin section. Another is that the structures of tissues, organelles, etc., are preserved better compared with the case of pre-embedding because post-embedding requires a few steps until embedding. However, post-embedding has a major disadvantage that antigens can be lost by dehydration or can be denatured by resin embedding, thus degrading the staining efficiency.

Related term
pre-embedding, immunoelectron microscopy

61. Skyrmion

Skyrmion is a vortex-like magnetic structure formed by the magnetic moments of electron spins. The term “Skymion” is named after Skyrme, a theoretical physicist of UK, who proposed the quantum structure. “Skymion” is also called “magnetic vortex” or “spin vortex”. Its size ranges from approximately several nm to 100 nm, depending on materials and chemical compositions. Skymions align like a two-dimensional crystal by cooling the specimen temperature and controlling the external magnetic field.

Skyrmion was first observed as a real-space image by Lorentz electron microscopy. Since then, it was also imaged by STEM using the DPC (differential phase contrast) method.

Courtesy of figures: Senior Researcher T. Matsumoto and Associate Professor N. Shibata The University of Tokyo (as of August 2015)

  • Skyrmion.jpg

Fig. (a) Schematic of the magnetic structure of skyrmions in a thin film. The directions of the magnetic moments of electron spins are indicated by arrows. The directions of those magnetic moments are perpendicular to the film plane at the core of the respective skyrmions, but swirl in the film plane at the peripheral region. Fig. (b) A real-space image of skymions acquired by the DPC (differential phase contrast) method (specimen: an Iron alloy). The colors in the figure depict the directions and magnitudes of the magnetic vectors in a two-dimensional plane.

Related term
Lorentz electron microscopy, differential phase contrast imaging