A wide range of preparation methods for TEM specimens are available, depending on the experimental requirements and material availability. These reach from the investigation of ground and powder materials on a support film to complex multi-step preparation procedures starting from suitable bulk material or thin films grown on a substrate. Until the advent of focused ion beam (FIB) devices, the standard technique in materials science was a combination of mechanical preparation and successive broad-beam milling using argon ions. However, mechanical polishing procedures to thin certain materials down to an atomically thin wedge are also available (88). Today, focused gallium or helium ions are used in FIB devices to prepare thin films of material, so-calledlamellae(100,101).
2.4 Specimen preparation
Some materials can be grown directly on electron-transparentmembranesand can even be structured using electron-beam lithography (102–104). Commercially available TEM membrane types include amorphous silicon nitride membranes with millimeter-scale lateral sizes and thicknesses down to 5 nm. These membranes have low internal stress, are smooth and chemically inert. For biological samples in cryo-TEM techniques, equally thin carbon films supported by copper or gold grids are a popular choice (88). Another route to TEM specimens for the case of layered materials with weak interlayer bonding is the exfoliation or “scotch-tape”
technique (105).
Ultramicrotomy is a related preparation technique originating from the biological sciences.
An ultramicrotome is a mechanical device that allows for cutting specimens into thin slices calledsections. For this purpose, a specimen block is mounted on an arm, that, in operation, moves downwards and slides the block along an atomically sharp knife edge (Fig.2.4A). The sections then float in a water trough until transferred onto a suitable specimen carrier (88). In contrast to most other TEM preparation methods, ultramicrotomy sections can reach lateral sizes of several hundreds of micrometers with very homogeneous thickness distributions (Fig.2.5).
In the preparation of material science specimens, ultramicrotomy also has its advantages as demonstrated in Chapters4and5. In this case, the typical fixation and dehydration procedures required for biological specimens are not necessary. Special resin and a mold are used to embed the material in a specimen block. The hardness of the resin should be as close as possible to that of the embedded material. If the specimen material is rigid enough, which holds true for
Knife edge
Figure 2.4: Specimen preparation using ultramicrotomy. (A) Schematic of the preparation process and the movement of the specimen arm. In operation, the arm moves downwards 1 and slides the specimen block along the atomically sharp edge of a diamond knife, resulting in an ultrathin section of the material. After cutting, the arm retracts slightly 2 , moves upwards 3 , feeds forward by the same length plus the desired section thickness 4 , and cuts the next section 1 . Ultrathin sections float on the water surface until transferred onto a specimen carrier. (B) Photograph of the diamond knife edge with a fresh section of graphite floating on the water surface taken through the binoculars of the ultramicrotome. The hexagonal bulk crystal is mounted on a block of gold and shown in the retracted position 3 .
A
500 µm
B C
100 µm
Figure 2.5: Light microscopy images of ultramicrotomed graphite films on a grid.(A) Two graphite films with 30 nm nominal thickness on a 200 lines per inch copper grid. (B) One of the graphite films observed in transmitted-light mode. The horizontal tear is due to a crystal defect of the bulk crystal. The field of view is indicated by the red box in A. See C for the scale bar. (C) The same graphite film as in B observed in reflected-light mode.
most (and even layered) crystalline materials, embedding can be completely abandoned and the material can be directly mounted on the specimen arm of the ultramicrotome as shown in Fig.2.4B (106).
Before sectioning, the cured specimen block must be trimmed in order to expose a small facet to the knife edge. Often, razor blades and glass knives are used for this purpose. After trimming, the exposed facet is sectioned in the ultramicrotome. Special ultramicrotomy diamond knives allow for section thicknesses even below 50 nm. For very thin bulk crystals, i.e., with a thickness of only a few tens to hundreds of microns, it may be worthwhile to glue the specimen to a block of soft material such as gold (Fig.2.4B). In case the complete crystal has been sectioned, the diamond knife will cut into soft gold instead of the stainless-steel specimen holder which prevents damage to the knife edge (106).
When floating in the water trough, individual sections can be carefully moved and manipulated using an eyelash hair. There are many different methods to retrieve the sections and place them on a support film or copper grid. One method is to use the eyelash to guide the section towards a TEM grid held slightly tilted under water. When gently rising the grid above the water level, the sections remain on the grid (106).
However, there are also other tools commercially available, such as the “perfect loop”. This tool essentially consists of a handle with a 3 mm ring attached. The ring can be used to lift a drop of water from the trough using the surface tension of the water, and with it the section that was floating on the water surface. When lowering the perfect loop onto a TEM grid positioned on a piece of filter paper, the water is absorbed by the paper, thus fixing the dried section on the grid.