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| title | chunk | source | category | tags | date_saved | instance |
|---|---|---|---|---|---|---|
| Transmission electron microscopy | 10/13 | https://en.wikipedia.org/wiki/Transmission_electron_microscopy | reference | science, encyclopedia | 2026-05-05T10:06:36.644553+00:00 | kb-cron |
Before sectioning, biological tissue is often embedded in an epoxy resin block and first trimmed using a razor blade into a trapezoidal block face. Thick sections are then cut from the block face. The thick sections are crudely stained with toluidine blue and examined for specimen and block orientation before thin sectioning. Biological tissue is then thinned to less than 100 nm on an ultramicrotome. The resin block is fractured as it passes over a glass or diamond knife edge. This method is used to obtain thin, minimally deformed samples that allow for the observation of tissue ultrastructure. Inorganic samples, such as aluminium, may also be embedded in resins and ultrathin sectioned in this way, using either coated glass, sapphire or larger angle diamond knives. To prevent charge build-up at the sample surface when viewing in the TEM, tissue samples need to be coated with a thin layer of conducting material, such as carbon.
=== Sample staining ===
TEM samples of biological tissues need high atomic number stains to enhance contrast. The stain absorbs the beam electrons or scatters part of the electron beam which otherwise is projected onto the imaging system. Compounds of heavy metals such as osmium, lead, uranium or gold (in immunogold labelling) may be used prior to TEM observation to selectively deposit electron dense atoms in or on the sample in desired cellular or protein region. This process requires an understanding of how heavy metals bind to specific biological tissues and cellular structures. Another form of sample staining is negative stain, where a larger amount of heavy metal stain is applied to the sample. The result is a sample with a dark background and the topological surface of the sample appearing lighter. Negative stain electron microscopy can be ideal for visualizing or forming 3D topological reconstructions of large proteins or macromolecular complexes (> 150 kDa). For smaller proteins, negative stain can be used as a screening step to find ideal sample concentration for cryogenic electron microscopy.
=== Mechanical milling === Mechanical polishing is also used to prepare samples for imaging on the TEM. Polishing needs to be done to a high quality, to ensure constant sample thickness across the region of interest. A diamond, or cubic boron nitride polishing compound may be used in the final stages of polishing to remove any scratches that may cause contrast fluctuations due to varying sample thickness. Even after careful mechanical milling, additional fine methods such as ion etching may be required to perform final stage thinning.
=== Chemical etching ===
Certain samples may be prepared by chemical etching, particularly metallic specimens. These samples are thinned using a chemical etchant, such as an acid, to prepare the sample for TEM observation. Devices to control the thinning process may allow the operator to control either the voltage or current passing through the specimen, and may include systems to detect when the sample has been thinned to a sufficient level of optical transparency.
=== Ion etching ===
Ion etching is a sputtering process that can remove very fine quantities of material. This is used to perform a finishing polish of specimens polished by other means. Ion etching uses an inert gas passed through an electric field to generate a plasma stream that is directed to the sample surface. Acceleration energies for gases such as argon are typically a few kilovolts. The sample may be rotated to promote even polishing of the sample surface. The sputtering rate of such methods is on the order of tens of micrometres per hour, limiting the method to only extremely fine polishing. Ion etching by argon gas has been recently shown to be able to file down MTJ stack structures to a specific layer which has then been atomically resolved. The TEM images taken in plan view rather than cross-section reveal that the MgO layer within MTJs contains a large number of grain boundaries that may be diminishing the properties of devices.
=== Ion milling (FIB) ===
More recently focused ion beam (FIB) methods have been used to prepare samples. FIB is a relatively new technique to prepare thin samples for TEM examination from larger specimens. Because FIB can be used to micro-machine samples very precisely, it is possible to mill very thin membranes from a specific area of interest in a sample, such as a semiconductor or metal. Unlike inert gas ion sputtering, FIB makes use of significantly more energetic gallium ions and may alter the composition or structure of the material through gallium implantation.
=== Nanowire assisted transfer === For a minimal introduction of stress and bending to transmission electron microscopy (TEM) samples (lamellae, thin films, and other mechanically and beam sensitive samples), when transferring inside a focused ion beam (FIB), flexible metallic nanowires can be attached to a typically rigid micromanipulator. The main advantages of this method include a significant reduction of sample preparation time (quick welding and cutting of nanowire at low beam current), and minimization of stress-induced bending, Pt contamination, and ion beam damage. This technique is particularly suitable for in situ electron microscopy sample preparation.
=== Replication ===
Samples may also be replicated using cellulose acetate film, the film subsequently coated with a heavy metal such as platinum, the original film dissolved away, and the replica imaged on the TEM. Variations of the replica technique are used for both materials and biological samples. In materials science a common use is for examining the fresh fracture surface of metal alloys.
== Modifications == The capabilities of the TEM can be further extended by additional stages and detectors, sometimes incorporated on the same microscope.
=== Scanning TEM ===