Figure 2: Schematics of the electron beam optical path in STEM. The beam is focused at the sample level, forming a probe that is scanned over the sample in a raster. The diameter of the probe (\(d\)) is determined by the convergence semi-angle (\(\alpha\)) and the wavelength of the electrons (\(\lambda\)) using the equation \(d=\lambda/\alpha\). The depth of field (\(\Delta z\)) is dependent on the convergence semi-angle (\(\alpha\)) and the wavelength of the electrons (\(\lambda\)) using the equation \(\Delta z=1.77\ \lambda/\alpha^{2}\). The camera length value can be modified to change the collection angles of the different detectors (there is a unique camera length for all the detectors).
In STEM, each horizontal line of the image is built point per point. The electron beam follows a raster scanning. To generate each pixel of the line, the number of electrons collected on the detector (BF, ADF or HAADF) is integrated at regular time intervals. The time interval is set by the experimenter and is called the dwell time, typically ranging between 1 and 4 µs in STEM of biological samples. In STEM, the pixel values are generated using amplitude contrast, which is proportional to the mass of the atoms. When the electron beam crosses light elements a great number of electrons pass through the sample without interaction and are collected by the BF detector. Conversely, when the electron beam crosses heavy elements, only a limited number of electrons pass through without interaction as most of these electrons are scattered away from the optical axis and are collected by the ADF or HAADF detector. The geometry of the beam (i.e., its convergence angle) determines the size of the probe and its depth of field. The smaller the convergence angle, the bigger the diameter of the probe. Moreover, as the depth of field is inversely proportional to the probe diameter, the smaller the convergence angle the greater the depth of field17. Thus, the size of the probe has a direct implication on the achievable resolution and must be matched with the image pixel size to avoid over- or under-sampling the specimen.