The CSTET technique could be optimized by working at higher voltage (300 kV
or higher), and by improving control over the illuminating probe beam. Another
area for optimization is on the detection side. A multi-ring annular detector that
would allow for multiple detection on a controllable range of acceptance angles
would allow for fine-tuning of contrast in the DF reconstructions. With improved
detectors, scan generators and electron optics, it may be possible to obtain excellent
results even from the thickest regions close to the nuclei of intact vitrified cells [93].
2.5 STEM Tomography Experimental Setup
at the Weizmann Institute
STEM tomography of plastic sections and CSTET are conducted on a Tecnai
F20 microscope operated at 200 kV, equipped with a HAADF detector at the 35 mm
port, and a Gatan BF/DF detector located under the fluorescent screen. Details of
the microscope configuration can be found online at http://www.weizmann.ac.il/
ChemicalResearchSupport/electron-microscopy/instrumentation. Switching between
TEM and STEM is done in a straightforward manner, by choosing the appropriate
“FEG Control” registers. Alignment after switching modes takes less than 15 min for
an experienced practitioner. The workflow, in brief is as follows:
2.5.1 CSTET data collection protocol
1. The protocol for sample vitrification of cells is identical to that for cryo-TEM.
2. The Gatan TAC100 retractable cryoblades and cold finger are cooled down at
least 20 min before inserting the sample. The EDS equipment on this instrument
precludes use of an FEI cryobox. The sample is inserted using standard routine
for side-entry holders.
3. In TEM or STEM imaging mode, at very low magnifications, appropriate areas
for study are selected, and those positions are saved.
4. The appropriate FEG register for STEM imaging is chosen, and a large condenser aperture is inserted for alignment. After basic alignments, an appropriate
small aperture is selected (10 or 20-micron apertures usually).
5. The dose is measured by imaging the beam (over vacuum) onto the Gatan
US4000 camera, situated under the fluorescent screen, and converting integrated
counts to electrons according to the calibrated value. The dose can be controlled
by the condenser aperture size, the spot size value, the extraction voltage, the
gun lens value, and scan rates. Typical dose values are 0.5–3 electrons/Å
2 /tilt.
6. CSTET datasets have been obtained with the FEI tomography software, but in
our current configuration, SerialEM [63] is used for data collection. The software is installed on a separate computer from the FEI microscope computer.
2 STEM Tomography in Biology
53
or higher), and by improving control over the illuminating probe beam. Another
area for optimization is on the detection side. A multi-ring annular detector that
would allow for multiple detection on a controllable range of acceptance angles
would allow for fine-tuning of contrast in the DF reconstructions. With improved
detectors, scan generators and electron optics, it may be possible to obtain excellent
results even from the thickest regions close to the nuclei of intact vitrified cells [93].
2.5 STEM Tomography Experimental Setup
at the Weizmann Institute
STEM tomography of plastic sections and CSTET are conducted on a Tecnai
F20 microscope operated at 200 kV, equipped with a HAADF detector at the 35 mm
port, and a Gatan BF/DF detector located under the fluorescent screen. Details of
the microscope configuration can be found online at http://www.weizmann.ac.il/
ChemicalResearchSupport/electron-microscopy/instrumentation. Switching between
TEM and STEM is done in a straightforward manner, by choosing the appropriate
“FEG Control” registers. Alignment after switching modes takes less than 15 min for
an experienced practitioner. The workflow, in brief is as follows:
2.5.1 CSTET data collection protocol
1. The protocol for sample vitrification of cells is identical to that for cryo-TEM.
2. The Gatan TAC100 retractable cryoblades and cold finger are cooled down at
least 20 min before inserting the sample. The EDS equipment on this instrument
precludes use of an FEI cryobox. The sample is inserted using standard routine
for side-entry holders.
3. In TEM or STEM imaging mode, at very low magnifications, appropriate areas
for study are selected, and those positions are saved.
4. The appropriate FEG register for STEM imaging is chosen, and a large condenser aperture is inserted for alignment. After basic alignments, an appropriate
small aperture is selected (10 or 20-micron apertures usually).
5. The dose is measured by imaging the beam (over vacuum) onto the Gatan
US4000 camera, situated under the fluorescent screen, and converting integrated
counts to electrons according to the calibrated value. The dose can be controlled
by the condenser aperture size, the spot size value, the extraction voltage, the
gun lens value, and scan rates. Typical dose values are 0.5–3 electrons/Å
2 /tilt.
6. CSTET datasets have been obtained with the FEI tomography software, but in
our current configuration, SerialEM [63] is used for data collection. The software is installed on a separate computer from the FEI microscope computer.
2 STEM Tomography in Biology
53