Transmission and scanning electron microscopy
Electron microscopes (EMs) use beams of electrons, exhibiting shorter wavelengths than visible light, for visualizing surfaces. The two types of EM are scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Both SEM and TEM require sample preparation, including immobilization. SEM has a much better depth of focus than light microscopy, with images formed by scanning the electron beam across the sample surface. For this technique, it is common to coat the sample surface with a thin metal film, which acts as an electron reflector and provides a path to the ground. In TEM a beam of electrons is passed through a very thin section of material, typically hundreds of nanometers in thickness. The detector displays where the electrons passed through the sample and where they did not. TEM sample preparation procedures can be time-consuming and highly specific for certain material types.
For example, organic materials require metallic stains such as uranyl acetate/lead citrate or potassium permanganate (Lewis and Knight, 1992). Alternative forms of EM have been developed to overcome issues related to sample preparation, such as dehydration. Cryogenic EM allows the samples to be frozen in a hydrated environment, thus mimicking the in vivo environment more closely (Carragher et al., 2015). Using this technique, Hendley et al. (2015) describe crystal formation and development of the different apatitic phases. However, freezing can also cause sample damage and therefore methods that avoid the freezing technique altogether have been developed. AirSEM allows samples to be imaged in air and does not require them to be coated (Han et al., 2015), while liquid-phase TEM allows imaging in a cell filled with liquid (Nielsen et al., 2014). Other advances in EM technology have led to the use of immu- nogold labeling, which allows specific proteins within the sample to be targeted (Mass et al., 2014) and the stitching of sequential TEM slices to render a 3-D image of the sample (Midgley and Weyland, 2003).
Selected-area electron diffraction (SAED) provides localized crystallographic information at high resolution and is performed inside EMs (Williams and Carter,
2009). Electrons pass through thin sample sections, their trajectories being altered by interactions with the atomic structure of the sample. The resultant diffraction pattern can be analyzed to determine the structure of the sample material. In conjunction with Raman spectroscopy, SAED has been used for the identification of calcite crystals isolated from the pineal gland (Baconnier et al., 2002).