extended dose tolerance exhibited by CSTET, dual-axis tomography schemes can
be considered using judicious dose-fractionation.
2.3.4 CSTET and Elemental Analysis of Bacteria
Much cryoTEM tomography (CET) has been performed on vitrified bacterial cells,
resulting in a wealth of information about bacterial cellular structure and function
(recent reviews: [77–80]). However, because of the limitations on specimen
thickness, most wildtype bacteria are too thick to study by CET. Lately, the use of
bacterial mini-cells [81] and ghosts [82] has become popular, because they are of
sufficiently thin dimensions to provide high-quality CET reconstructions.
Since CSTET allows for tomography of specimens around three times thicker than
for CET (Sect. 2.2.1), a wide variety of cells can be observed without the need for
sectioning or use of mini-cells.
Comparison of CSTET and CET reconstructions of Agrobacteria tumefaciens
revealed that similar features could be found with CSTET, but with much improved
contrast over noise (Fig. 2.5).
As was shown previously for thick plastic sections [32] (Fig. 2.4), we found that
useful data could be collected up to very high tilts with the CSTET mode, while for
TEM imaging, the data collected at high tilts did not provide information that
improved the reconstructions. This was measured by comparing cross-correlations
of odd-even divided datasets. Only for the CSTET data did addition of high-tilt data
increase the coefficient value (supplementary Fig. 5 in [20]), indicating a contribution of added information above noise.
T-phage attack on an E coli cell is observable in three dimensions by CSTET,
even though the overall thickness of the sample exceeds 1 micron (Fig. 2.6). The
phages can be observed in early stages of attachment and cargo delivery (unpublished results).
As described in Sect. 2.2.4, analytical measurements by EDS or EELS are
straightforward in STEM mode. Unlike the approach of freeze-drying the entire
grid, the area of interest can be targeted exclusively in STEM while preserving the
vitrified state of the rest of the sample. Thus, for instance, sample thickness can be
directly calculated with EELS measurements [83]. In addition, elemental analysis
was performed by EDS on the dense bodies found in vitrified Agrobacteria
tumefaciens, and found to contain excess phosphorus (see Fig. 2.7), identifying
them as polyphosphate bodies (PPBs) [84]. Because elastic scattering is directly
related to atomic number (Fig. 2.2), it was also possible, by judicious choice of
collection angle, to map and quantify the distribution of phosphorus in the DF
image [84]. Such Z contrast can of course be generalized to other situations where
the elemental composition is known.
2 STEM Tomography in Biology
47
be considered using judicious dose-fractionation.
2.3.4 CSTET and Elemental Analysis of Bacteria
Much cryoTEM tomography (CET) has been performed on vitrified bacterial cells,
resulting in a wealth of information about bacterial cellular structure and function
(recent reviews: [77–80]). However, because of the limitations on specimen
thickness, most wildtype bacteria are too thick to study by CET. Lately, the use of
bacterial mini-cells [81] and ghosts [82] has become popular, because they are of
sufficiently thin dimensions to provide high-quality CET reconstructions.
Since CSTET allows for tomography of specimens around three times thicker than
for CET (Sect. 2.2.1), a wide variety of cells can be observed without the need for
sectioning or use of mini-cells.
Comparison of CSTET and CET reconstructions of Agrobacteria tumefaciens
revealed that similar features could be found with CSTET, but with much improved
contrast over noise (Fig. 2.5).
As was shown previously for thick plastic sections [32] (Fig. 2.4), we found that
useful data could be collected up to very high tilts with the CSTET mode, while for
TEM imaging, the data collected at high tilts did not provide information that
improved the reconstructions. This was measured by comparing cross-correlations
of odd-even divided datasets. Only for the CSTET data did addition of high-tilt data
increase the coefficient value (supplementary Fig. 5 in [20]), indicating a contribution of added information above noise.
T-phage attack on an E coli cell is observable in three dimensions by CSTET,
even though the overall thickness of the sample exceeds 1 micron (Fig. 2.6). The
phages can be observed in early stages of attachment and cargo delivery (unpublished results).
As described in Sect. 2.2.4, analytical measurements by EDS or EELS are
straightforward in STEM mode. Unlike the approach of freeze-drying the entire
grid, the area of interest can be targeted exclusively in STEM while preserving the
vitrified state of the rest of the sample. Thus, for instance, sample thickness can be
directly calculated with EELS measurements [83]. In addition, elemental analysis
was performed by EDS on the dense bodies found in vitrified Agrobacteria
tumefaciens, and found to contain excess phosphorus (see Fig. 2.7), identifying
them as polyphosphate bodies (PPBs) [84]. Because elastic scattering is directly
related to atomic number (Fig. 2.2), it was also possible, by judicious choice of
collection angle, to map and quantify the distribution of phosphorus in the DF
image [84]. Such Z contrast can of course be generalized to other situations where
the elemental composition is known.
2 STEM Tomography in Biology
47