3.4.2 Data Acquisition Software
The user has a choice in data collection software packages, and the needs of the
project will dictate which is used. As it is critically important to minimize exposure of
the specimen to the electron beam prior to data collection, data collection is performed under the ‘low dose’ philosophy. This means that an area of the grid is imaged
once and stored digitally together with metadata about which part of the grid the
image corresponds to, and its magnification. The user can then perform additional
targeting ‘offline’ by referring back to this image instead of re-exposing the area to the
electron beam. In general the software will use a number of preset magnifications to
first take an overview grid ‘atlas’. Favourable squares can be identified on the grid
atlas and imaged at higher magnification, and targets selected from the stored square
images. Because electron dosages per unit area are lower for low magnification
images, the total dose before data collection is essentially negligible.
A variety of sophisticated data collection software packages integrate this philosophy into their design. In practice, tilt series collection is not straightforwards, as
optic stability, stage accuracy and reproducibility, and specimen stability may not be
ideal, requiring constant monitoring and compensation. Data acquisition software
therefore utilizes a low dose philosophy and controls microscope optics, stage
movement, and imaging, automatically applying entire sets of presets to simultaneously change parameters such as image shifts and beam intensity together with
magnification changes while tilt series images are being collected. Although the end
goal is the same—one or more tilt series of the specimen—there is an array of software
to acquire data (at the time of writing the “Software Tools for Molecular Microscopy”
Wikibook provided an up-to-date list). Proprietary solutions exist; at the time of
writing JEOL produces TEMography™ for JEOL microscopes; FEI Company produces the Xplore3D™ Tomography Suite and its successor, Tomography 4.0™, for
FEI microscopes; TVIPS GmbH produces EM-Tomo for TVIPS cameras attached to
a variety of microscopes; and Gatan Inc. produces a tomography module for their
GMS imaging suite for microscopes fitted with Gatan cameras.
The user, however, also has a number of free software options, the choice resting
upon the desired balance between interactivity and automation, and ease of
installation. At one end of the spectrum, tilt series can be collected manually by the
user without dedicated software, ensuring the specimen remains at eucentric height.
In practice this approach is rarely followed, except in exceptional circumstances
where user intervention is essential, for example with manual alignment of phase
plates [44]. Arguably the most widely used package is SerialEM [45] (a sister
package to the IMOD suite of programs for tomographic reconstruction, manipulation, and analysis), best known for its flexibility, customizability, and
platform-agnosticism, and batch tilt series acquisition mode. Another free solution
to tilt series acquisition is UCSF Tomo [46] which is considerably simpler in
presentation than SerialEM and employs a prediction-based scheme to maintain
specimen focus and centring in the viewing window for batch tomography mode
that is sufficiently robust in terms of targeting and tracking for routine overnight tilt
3 Electron Cryo-Tomography
71
The user has a choice in data collection software packages, and the needs of the
project will dictate which is used. As it is critically important to minimize exposure of
the specimen to the electron beam prior to data collection, data collection is performed under the ‘low dose’ philosophy. This means that an area of the grid is imaged
once and stored digitally together with metadata about which part of the grid the
image corresponds to, and its magnification. The user can then perform additional
targeting ‘offline’ by referring back to this image instead of re-exposing the area to the
electron beam. In general the software will use a number of preset magnifications to
first take an overview grid ‘atlas’. Favourable squares can be identified on the grid
atlas and imaged at higher magnification, and targets selected from the stored square
images. Because electron dosages per unit area are lower for low magnification
images, the total dose before data collection is essentially negligible.
A variety of sophisticated data collection software packages integrate this philosophy into their design. In practice, tilt series collection is not straightforwards, as
optic stability, stage accuracy and reproducibility, and specimen stability may not be
ideal, requiring constant monitoring and compensation. Data acquisition software
therefore utilizes a low dose philosophy and controls microscope optics, stage
movement, and imaging, automatically applying entire sets of presets to simultaneously change parameters such as image shifts and beam intensity together with
magnification changes while tilt series images are being collected. Although the end
goal is the same—one or more tilt series of the specimen—there is an array of software
to acquire data (at the time of writing the “Software Tools for Molecular Microscopy”
Wikibook provided an up-to-date list). Proprietary solutions exist; at the time of
writing JEOL produces TEMography™ for JEOL microscopes; FEI Company produces the Xplore3D™ Tomography Suite and its successor, Tomography 4.0™, for
FEI microscopes; TVIPS GmbH produces EM-Tomo for TVIPS cameras attached to
a variety of microscopes; and Gatan Inc. produces a tomography module for their
GMS imaging suite for microscopes fitted with Gatan cameras.
The user, however, also has a number of free software options, the choice resting
upon the desired balance between interactivity and automation, and ease of
installation. At one end of the spectrum, tilt series can be collected manually by the
user without dedicated software, ensuring the specimen remains at eucentric height.
In practice this approach is rarely followed, except in exceptional circumstances
where user intervention is essential, for example with manual alignment of phase
plates [44]. Arguably the most widely used package is SerialEM [45] (a sister
package to the IMOD suite of programs for tomographic reconstruction, manipulation, and analysis), best known for its flexibility, customizability, and
platform-agnosticism, and batch tilt series acquisition mode. Another free solution
to tilt series acquisition is UCSF Tomo [46] which is considerably simpler in
presentation than SerialEM and employs a prediction-based scheme to maintain
specimen focus and centring in the viewing window for batch tomography mode
that is sufficiently robust in terms of targeting and tracking for routine overnight tilt
3 Electron Cryo-Tomography
71