variations in the scattering of electrons that are produced by spatial modulations in
density and differential scattering cross-sections, integrated over a range from inner
to outer cutoff angles of the detector. Note that when the specimen is removed the
ADF signal is zero. A bright field (BF) detector is configured to match, more or
less, the illuminating cone that diverges from the focus at the specimen. Scattering
to angles beyond this cone causes a loss in signal and therefore dark contrast on a
brighter background. Since the BF detector collects only those electrons that deviate
little from direct transmission, the BF image ideally reflects the variation in total
scattering from point to point. For a given chemical composition, the DF signal is
directly proportional to the number of scattering atoms, i.e., the mass density. Mass
measurement by STEM [9, 10, 11, 14, 25] have therefore frequently been used to
access information not directly accessible by other techniques.
The DF signal at larger scattering angles is comprised of elastically scattered
electrons. These may undergo additional inelastic scattering, but inelastic scattering
alone will not produce a signal at high angles. The differential scattering crosssection for elastic scattering (2.1, 2.2) follows the simple Rutherford dependence of
Z
2 at high scattering angles where screening effects are negligible. Because heavy
atom stains scatter electrons elastically, several orders of magnitude more strongly
than the light atoms of biological matter, the stain in plastic sections provides most
of the signal in DF images that only collect the high-angle scattering. According to
Fig. 2.2, an inner collection angle of several 10s of mrad is typically enough to
suppress the scattering of the matrix of light elements and to provide a good
signal-to-noise-ratio for dark-field images of stained samples.
For plastic sections, the BF contrast peaks at stained areas where electrons are
scattered away from the forward direction, i.e., these electrons are not collected by
the BF detector. The ADF detects primarily these stained areas but without the
background of transmitted or forward scattered electrons that are present in the BF.
For cryo-preserved specimens, objects in the cells that have higher concentrations
of heavy atoms will be preferentially detected by ADF, while a combination of
mass/thickness contrast and variations in atomic composition provide the BF signal.
As will be discussed in the following Sect. 2.2.3, for thick specimens in tomography applications, these advantages of background-free DF imaging are offset due
to beam divergence and multiple scattering through the long sample path, and
therefore BF images are often used for plastic thick samples.
2.2.3 Resolution and Thick Sample Effects
Inelastic and plural scattering in a thick specimen lowers the tomographic resolution
[26]. In most biological specimens the interest is in structural details much larger
than a single atom (except for the case of single-particle reconstructions of purified
macromolecules). In such a case plural electron scattering in thick samples may be
tolerated to an extent that preserves the three-dimensional structure, even if atomic
2 STEM Tomography in Biology
39
density and differential scattering cross-sections, integrated over a range from inner
to outer cutoff angles of the detector. Note that when the specimen is removed the
ADF signal is zero. A bright field (BF) detector is configured to match, more or
less, the illuminating cone that diverges from the focus at the specimen. Scattering
to angles beyond this cone causes a loss in signal and therefore dark contrast on a
brighter background. Since the BF detector collects only those electrons that deviate
little from direct transmission, the BF image ideally reflects the variation in total
scattering from point to point. For a given chemical composition, the DF signal is
directly proportional to the number of scattering atoms, i.e., the mass density. Mass
measurement by STEM [9, 10, 11, 14, 25] have therefore frequently been used to
access information not directly accessible by other techniques.
The DF signal at larger scattering angles is comprised of elastically scattered
electrons. These may undergo additional inelastic scattering, but inelastic scattering
alone will not produce a signal at high angles. The differential scattering crosssection for elastic scattering (2.1, 2.2) follows the simple Rutherford dependence of
Z
2 at high scattering angles where screening effects are negligible. Because heavy
atom stains scatter electrons elastically, several orders of magnitude more strongly
than the light atoms of biological matter, the stain in plastic sections provides most
of the signal in DF images that only collect the high-angle scattering. According to
Fig. 2.2, an inner collection angle of several 10s of mrad is typically enough to
suppress the scattering of the matrix of light elements and to provide a good
signal-to-noise-ratio for dark-field images of stained samples.
For plastic sections, the BF contrast peaks at stained areas where electrons are
scattered away from the forward direction, i.e., these electrons are not collected by
the BF detector. The ADF detects primarily these stained areas but without the
background of transmitted or forward scattered electrons that are present in the BF.
For cryo-preserved specimens, objects in the cells that have higher concentrations
of heavy atoms will be preferentially detected by ADF, while a combination of
mass/thickness contrast and variations in atomic composition provide the BF signal.
As will be discussed in the following Sect. 2.2.3, for thick specimens in tomography applications, these advantages of background-free DF imaging are offset due
to beam divergence and multiple scattering through the long sample path, and
therefore BF images are often used for plastic thick samples.
2.2.3 Resolution and Thick Sample Effects
Inelastic and plural scattering in a thick specimen lowers the tomographic resolution
[26]. In most biological specimens the interest is in structural details much larger
than a single atom (except for the case of single-particle reconstructions of purified
macromolecules). In such a case plural electron scattering in thick samples may be
tolerated to an extent that preserves the three-dimensional structure, even if atomic
2 STEM Tomography in Biology
39