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.