conveniently with the pixel dimensions and the pixel dwell time. In parallel, EDS
can be used to analyze characteristic X-rays generated in the interaction volume on
the nano-scale.
In principle, STEM EELS and EDS signals can be recorded with a similar
resolution to the bright-field STEM signal; the ultimate spatial resolution is limited
by the broadening of the beam. For EDS, multiple inelastic scattering also degrades
the spatial resolution, since it results in the production of fast secondary electrons
that generate characteristic x-rays, particularly from light elements. However, the
cross-sections for inner-shell excitation (“core-loss”) and a subsequent X-ray
emission are typically 4–6 orders of magnitude smaller than a cross-section for
elastic scattering or plasmon scattering [21]. As a result, the practically meaningful
resolution for elemental analysis becomes dose-limited and considerably worse than
the attainable resolution in a bright-field image.
Despite the fact that the resolution becomes dose limited, analytical signals
observed in STEM have clear and compelling applications for biological samples.
Whole-cell elemental mapping has demonstrated compositional distribution in
freeze-dried biological cells via EDS in STEM [38]. Recent advancement in the
collection angle of EDS detectors enhances the possibilities of chemical analysis
and mapping of biological specimens [39]. The complementary approach of EELS
was employed to identify light atoms (e.g., C, N, O, P, S) and light or transition
metals (e.g., Ca, Mg, Zn, Cu, Fe) in thin sections [8, 19, 38, 39].
2.3 STEM Tomography
2.3.1 Historical Background
During the first decades since the invention of the electron microscope, most of the
information in structural cell biology at submicron dimensions was derived from
bright field TEM observations of thin-sectioned plastic-embedded specimens. The
thickness limitation was partially circumvented by the imaging of consecutive
(serial) sections, which allowed for 3D reconstruction from many 2D slices [40].
High acceleration voltage TEMs (HVTEM), which became available towards the
end of the 1960s, are superior to low voltage TEMs with respect to penetration
power, lens aberrations and electron-beam damage, see (2.1, 2.2). They were thus
used for the observation of sections between 0.5 to several microns in thickness [41,
42]. Micrographs of such observations were difficult or impossible to interpret, since
much information was lost when the complex 3D structure contained in the section
was projected onto a 2D negative. Thus stereo pairs were obtained in order to regain
and analyze some 3D information [41, 43– 45], and later electron tomography was
developed in order to obtain 3D reconstructions [46]. While HVTEM instruments
have higher penetration power than conventional TEM, they still suffer from lens
2 STEM Tomography in Biology
43
can be used to analyze characteristic X-rays generated in the interaction volume on
the nano-scale.
In principle, STEM EELS and EDS signals can be recorded with a similar
resolution to the bright-field STEM signal; the ultimate spatial resolution is limited
by the broadening of the beam. For EDS, multiple inelastic scattering also degrades
the spatial resolution, since it results in the production of fast secondary electrons
that generate characteristic x-rays, particularly from light elements. However, the
cross-sections for inner-shell excitation (“core-loss”) and a subsequent X-ray
emission are typically 4–6 orders of magnitude smaller than a cross-section for
elastic scattering or plasmon scattering [21]. As a result, the practically meaningful
resolution for elemental analysis becomes dose-limited and considerably worse than
the attainable resolution in a bright-field image.
Despite the fact that the resolution becomes dose limited, analytical signals
observed in STEM have clear and compelling applications for biological samples.
Whole-cell elemental mapping has demonstrated compositional distribution in
freeze-dried biological cells via EDS in STEM [38]. Recent advancement in the
collection angle of EDS detectors enhances the possibilities of chemical analysis
and mapping of biological specimens [39]. The complementary approach of EELS
was employed to identify light atoms (e.g., C, N, O, P, S) and light or transition
metals (e.g., Ca, Mg, Zn, Cu, Fe) in thin sections [8, 19, 38, 39].
2.3 STEM Tomography
2.3.1 Historical Background
During the first decades since the invention of the electron microscope, most of the
information in structural cell biology at submicron dimensions was derived from
bright field TEM observations of thin-sectioned plastic-embedded specimens. The
thickness limitation was partially circumvented by the imaging of consecutive
(serial) sections, which allowed for 3D reconstruction from many 2D slices [40].
High acceleration voltage TEMs (HVTEM), which became available towards the
end of the 1960s, are superior to low voltage TEMs with respect to penetration
power, lens aberrations and electron-beam damage, see (2.1, 2.2). They were thus
used for the observation of sections between 0.5 to several microns in thickness [41,
42]. Micrographs of such observations were difficult or impossible to interpret, since
much information was lost when the complex 3D structure contained in the section
was projected onto a 2D negative. Thus stereo pairs were obtained in order to regain
and analyze some 3D information [41, 43– 45], and later electron tomography was
developed in order to obtain 3D reconstructions [46]. While HVTEM instruments
have higher penetration power than conventional TEM, they still suffer from lens
2 STEM Tomography in Biology
43