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.