Transmission electron microscopy (TEM) – imaging

About this technique

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Transmission electron microscopy (TEM) generates images by passing a beam of electrons through a specimen. The electron beam must be able penetrate the specimen in order to create a transmission (shadow) image. Samples must therefore be very thin and are prepared either but cutting extremely thin sections or, for solutions or suspensions or macromolecules, viruses or nanoparticles, deposited directly onto support films.

TEM specimen thicknesses are typically less than 100 nm. For biological materials, specimen preparation usually entails: fixation to preserve the structure; resin impregnation to replace the water in the specimen and make it rigid; staining, usually with heavy metal ions, to produce differential contrast between structures, which take up the stain to differing degrees. Immunogold labeling can be used identify areas where specific biologically important molecules are located and this can be correlated with optical microscopy techniques (covered in the correlative microscopy section); finally, ultramicrotomy is used to cut thin sections. Some TEMs have the ability to obtain tilt sequences to enable the three dimensional structure to be determined via tomography. This is dealt with in its own section.

For materials work, a very wide range of techniques is available for producing electron transparent specimens. Electrochemical jet polishing is widely used for thinning conductive metal specimens. Focused ion beam (FIB) milling is a versatile technique that enables region-specific TEM specimens to be fabricated from most inorganic materials. Broad ion beam techniques also find use in thinning and cleaning ceramic, metallic and semiconductor materials. Polymeric materials are prepared much like biological materials and are often heavy metal stained then sectioned using ultramicrotomy at low temperatures.

As TEMs operate under very high vacuum, specimens must be vacuum and electron beam compatible (i.e. dry). For soft matter that is dependent on solvent for its structure, such as liposomes, cryo-TEM techniques may be required. Here a sample of the solvent containing the structure of interest is plunge frozen to preserve the microstructure within a membrane of frozen amorphous ice. This is transferred to the microscope while still frozen and the material is imaged using the amorphous ice as a support film.

Even basic TEMs are usually capable of magnifying features several hundred thousand times, and so structural details at the nanometre scale are readily resolved. TEM imaging finds widespread use in characterising such things as biological ultrastructure, nanoparticles, thin films, crystalline materials containing fine-scale precipitates/defects etc. High performance TEM can resolve atomic structure in crystalline materials. Electron diffraction patterns can be obtained from features of interest to understand their crystal structure/orientation relationship. On instruments equipped with X-ray or electron energy loss spectrometers compositional analysis/mapping is also possible. Scanning TEM capabilities further extend the range of imaging and analysis modalities available.