2D scanning transmission electron microscopy approaches for parasitology
STEM principle & applications
Compared to TEM which uses a spread electron beam, STEM uses a focused electron beam forming a probe3 which can reach sub-nanometre diameter. This probe is scanned at the specimen level and the transmitted electrons are collected by different post-specimen detectors. Depending on the angle at which electrons have been scattered after their interaction with the sample, electrons are recorded by either bright field (BF), annular dark field (ADF), or high angle annular dark field detectors (HAADF) (Fig. 2). Electrons that do not interact with the sample are not scattered and are collected by the BF detector. Electrons interacting inelastically with the sample (interaction with the electron cloud) are scattered at relatively small angles and are typically collected by the ADF detector. Finally, electrons that interact with the atomic nucleus undergo a scattering at high angle and are collected by the HAADF detector. It is important to note that an electron that went through multiple inelastic scattering has some probability to exit the sample at high angle and then be detected by the HAADF. However, this multiple successive inelastic scattering can only exist in samples with a thickness equal to several times the inelastic mean free path of the electron (i.e., biological samples which are thicker than 0.5 µm). The distance between the specimen and the detectors is called the camera length. It can be virtually modified using lenses of the microscope, thus allowing to vary the collection angles of the different detectors. Inelastic scattering can be discarded by choosing the right camera length and generating images based solely on electrons collected by the BF detector. This method is referred to as STEM BF, which is the mode that will be mainly presented in this review.