aberrations as well as from small depth of focus, resulting in blurred images especially at high tilt angles when tilt series are acquired for electron tomography
reconstruction [47, 48].
The notion that STEM imaging is more suitable for the observation of thick
biological sections than conventional TEM (CTEM) was known already in 1938
when von Ardenne built the first STEM: “At that time this type (i.e. STEM) had
priority for us because there was a chance that, even in investigations of relatively
thick specimens, such as standard microtome sections, chromatic aberration could
be kept low” [49]. After a long gap in the advance of STEM technology, Crewe and
his colleagues revived the field and constructed the modern STEM in the 1960s.
Crewe & Groves stated that the penetration power of a microscope is not only the
ability of a microscope to penetrate through the sample, but also “the ability of a
given microscope to produce a high quality image of a thick object” [50]. In this
respect the STEM performs better than CTEM as “For a carbon specimen one
micron thick a STEM operating in bright field at 90 keV produces an image which
is roughly equivalent to that of a CEM operating in bright field at 1 MeV” [50]. It is
therefore intriguing to note that the advantage of STEM in the observation of thick
biological sections was only picked up for further development decades later,
thanks to a small community that optimized STEM biological imaging [8]. STEM
was used in a biological context mainly as an analytical tool—for spectroscopy and
elemental mapping using EDS or EELS [7, 8, 18, 19, 51, 52], for mass measurements of proteins [10, 12, 13], or for the detection of heavy atom particles which
were either naturally occurring in the sample or added as probes or labels [14, 53–
55]. Much work was conducted over the years to characterize and optimize imaging
conditions of thick samples in the STEM, which paved the way to STEM
tomography of such samples. Gentsch et al. [56] investigated the “top bottom”
effect (see Sect. 2.2.3). Smith and Cowley investigated the calculated dependence
of image contrast on the collection angle of the STEM detector [57]. They corroborated their calculations with measured contrast of sections up to 2 lm in
thickness. They also advocated the use of energy filters for contrast enhancement of
thick sections. Indeed, Colliex et al. [7] used a dedicated STEM equipped with an
energy loss spectrometer to find optimal conditions for visualizing thick sections
with selected inelastically scattered electrons.
Beorchia et al. [58] used a 300 kV STEM explicitly with the aim of optimizing
STEM imaging of thick sections for tomographic reconstruction. To that end they
used plastic sections with thickness ranging between 0.5 and 8 lm. They calculated
the rate and degree of section shrinkage upon electron irradiation by measuring the
distance between gold beads adhered to both sides of the sections and found that
sections shrink between 45% for a 1 lm thick section to 30% for an 8 lm thick
sections. They obtained tilt series ranging between ± 50° with images having sharp
contrast and good focus.
44
S. G. Wolf et al.
reconstruction [47, 48].
The notion that STEM imaging is more suitable for the observation of thick
biological sections than conventional TEM (CTEM) was known already in 1938
when von Ardenne built the first STEM: “At that time this type (i.e. STEM) had
priority for us because there was a chance that, even in investigations of relatively
thick specimens, such as standard microtome sections, chromatic aberration could
be kept low” [49]. After a long gap in the advance of STEM technology, Crewe and
his colleagues revived the field and constructed the modern STEM in the 1960s.
Crewe & Groves stated that the penetration power of a microscope is not only the
ability of a microscope to penetrate through the sample, but also “the ability of a
given microscope to produce a high quality image of a thick object” [50]. In this
respect the STEM performs better than CTEM as “For a carbon specimen one
micron thick a STEM operating in bright field at 90 keV produces an image which
is roughly equivalent to that of a CEM operating in bright field at 1 MeV” [50]. It is
therefore intriguing to note that the advantage of STEM in the observation of thick
biological sections was only picked up for further development decades later,
thanks to a small community that optimized STEM biological imaging [8]. STEM
was used in a biological context mainly as an analytical tool—for spectroscopy and
elemental mapping using EDS or EELS [7, 8, 18, 19, 51, 52], for mass measurements of proteins [10, 12, 13], or for the detection of heavy atom particles which
were either naturally occurring in the sample or added as probes or labels [14, 53–
55]. Much work was conducted over the years to characterize and optimize imaging
conditions of thick samples in the STEM, which paved the way to STEM
tomography of such samples. Gentsch et al. [56] investigated the “top bottom”
effect (see Sect. 2.2.3). Smith and Cowley investigated the calculated dependence
of image contrast on the collection angle of the STEM detector [57]. They corroborated their calculations with measured contrast of sections up to 2 lm in
thickness. They also advocated the use of energy filters for contrast enhancement of
thick sections. Indeed, Colliex et al. [7] used a dedicated STEM equipped with an
energy loss spectrometer to find optimal conditions for visualizing thick sections
with selected inelastically scattered electrons.
Beorchia et al. [58] used a 300 kV STEM explicitly with the aim of optimizing
STEM imaging of thick sections for tomographic reconstruction. To that end they
used plastic sections with thickness ranging between 0.5 and 8 lm. They calculated
the rate and degree of section shrinkage upon electron irradiation by measuring the
distance between gold beads adhered to both sides of the sections and found that
sections shrink between 45% for a 1 lm thick section to 30% for an 8 lm thick
sections. They obtained tilt series ranging between ± 50° with images having sharp
contrast and good focus.
44
S. G. Wolf et al.