STEM image-formation is an incoherent process, and the much-discussed issue of
chromatic aberration in TEM imaging is irrelevant. For phase-contrast TEM imaging,
only elastically scattered coherent electrons provide a useful signal. Inelastically
scattered electrons cause damage to the sample and add blur to the image. For
tomography they are often removed with an energy filter. Incoherent STEM imaging
is less affected by inelastic scattering and thus STEM imaging can be very efficient,
especially for thick samples where phase coherence is lost. Finally, because the beam
is rastered, dynamic focusing at high tilt is naturally available for tilt series recording
in tomography. Dynamic focusing is simply achieved by adjusting the focus for each
line during the scan,
1 according to its geometric location on the tilted sample.
In the following sections, we will describe STEM image formation and complementary analytical tools, and provide a framework for understanding the
advantages of STEM imaging for tomography. Afterwards, we will discuss applications of STEM in tomography of thick biological specimens, first for plasticembedded samples, and then for cryo-preserved specimens.
2.2 Basics of STEM Imaging of Biological Material
2.2.1 Electron-Sample Interactions
Contrast and resolution of STEM and TEM images are affected by the interaction of
the electron beam with the sample. The primary interactions between the fast
electron and the atoms in the sample are due to electrostatic Coulomb forces. Elastic
and inelastic, as well as coherent and incoherent processes occur in the scattering of
electrons [18, 21]. Elastic scattering can result in a rather large deflection of the
incident electron but does not change the state of a target atom; specifically, no
energy is lost by the electron. Quasi-elastic scattering is connected with oscillations
in molecules or phonon excitations that attenuates the coherence and leads to thermal
diffuse scattering. Inelastic events (wherein electrons do lose energy after interaction
with the specimen) originate from interaction between the incident electron and the
target electrons by excitation of collective oscillations (plasmons), valence electrons,
or the ionization of inner atomic shells. Inelastically scattered electrons appear as a
new source within the specimen and attenuate or destroy coherent interference
effects. In addition the deposited energy causes damage to the sample.
Both the angular scattering and the total scattering amplitude depend strongly on
the nuclear charge and thus on the atomic number Z of the atoms in the sample.
This dependence is utilized in the various methods associated with electron
microscopy imaging and spectroscopy. In a crystalline solid, diffraction effects
change the angular dependence of scattering dramatically, but in amorphous solids,
1
The focus is adjusted per line, assuming that the scan lines are parallel to the tilt axis.
2 STEM Tomography in Biology
35
chromatic aberration in TEM imaging is irrelevant. For phase-contrast TEM imaging,
only elastically scattered coherent electrons provide a useful signal. Inelastically
scattered electrons cause damage to the sample and add blur to the image. For
tomography they are often removed with an energy filter. Incoherent STEM imaging
is less affected by inelastic scattering and thus STEM imaging can be very efficient,
especially for thick samples where phase coherence is lost. Finally, because the beam
is rastered, dynamic focusing at high tilt is naturally available for tilt series recording
in tomography. Dynamic focusing is simply achieved by adjusting the focus for each
line during the scan,
1 according to its geometric location on the tilted sample.
In the following sections, we will describe STEM image formation and complementary analytical tools, and provide a framework for understanding the
advantages of STEM imaging for tomography. Afterwards, we will discuss applications of STEM in tomography of thick biological specimens, first for plasticembedded samples, and then for cryo-preserved specimens.
2.2 Basics of STEM Imaging of Biological Material
2.2.1 Electron-Sample Interactions
Contrast and resolution of STEM and TEM images are affected by the interaction of
the electron beam with the sample. The primary interactions between the fast
electron and the atoms in the sample are due to electrostatic Coulomb forces. Elastic
and inelastic, as well as coherent and incoherent processes occur in the scattering of
electrons [18, 21]. Elastic scattering can result in a rather large deflection of the
incident electron but does not change the state of a target atom; specifically, no
energy is lost by the electron. Quasi-elastic scattering is connected with oscillations
in molecules or phonon excitations that attenuates the coherence and leads to thermal
diffuse scattering. Inelastic events (wherein electrons do lose energy after interaction
with the specimen) originate from interaction between the incident electron and the
target electrons by excitation of collective oscillations (plasmons), valence electrons,
or the ionization of inner atomic shells. Inelastically scattered electrons appear as a
new source within the specimen and attenuate or destroy coherent interference
effects. In addition the deposited energy causes damage to the sample.
Both the angular scattering and the total scattering amplitude depend strongly on
the nuclear charge and thus on the atomic number Z of the atoms in the sample.
This dependence is utilized in the various methods associated with electron
microscopy imaging and spectroscopy. In a crystalline solid, diffraction effects
change the angular dependence of scattering dramatically, but in amorphous solids,
1
The focus is adjusted per line, assuming that the scan lines are parallel to the tilt axis.
2 STEM Tomography in Biology
35