the sample is a limiting factor. During the first few hours of the run, the stability
needs to be monitored but with as minimal disturbance to the microscope as possible. If the dwell time necessary to achieve uniform milling is too low to achieve a
good SNR, reduce the FOV or increase the pixel size. It may also be necessary to
increase the FIB beam current or adjust the milling time by correcting the milling
rates and/or increasing the milling depth. Image acquisition and milling times
should be defined in relation to specimen drift [34]. There is always a balance
between these factors since the series of SEM images is collected automatically. If
the acquisition time is too long, the sample may either drift into the FIB beam thus
increasing the slice thickness or drift away from the FIB beam so that the surface is
burned by the imaging SEM beam and not milled away. As Maco et al. [75]
instructs, “any interruption to the imaging procedure will cause irregular thicknesses to be removed by the milling beam”. When parameters that produce quality
data with uniform milling are found it is crucial to minimise disturbances, with only
brief checks of focus and astigmatism [75].
5.5 Discussion
Volume SEM utilising the SBEM and FIB-SEM enables better appreciation of the
complexity of biological systems, which by nature are in three dimensions. They
produce a series of 2D images that are combined to create a 3D volume at the
nanoscale. This new generation of techniques has taken serial sectioning into a new
realm. It is now possible to understand the relative relationship between different
structures in a full volume rather than as individual two-dimensional images that are
obtained from routine TEM of ultrathin sections. Despite the existence of ssTEM
for more than 60 years, this method is limited by its laborious nature. With the
availability of commercial instruments that allow for automation, SBEM and
FIB-SEM have received wide acceptance in the scientific community and have now
made volume EM a routine technique.
While SBEM and FIB-SEM lack the ultimate resolution of a TEM, they fill a
major gap in resolution between light microscopy and the high end TEM. Many
biological studies do not require the highest resolution in order to answer the
scientific question being asked. A resolution of 5 Â 5 Â 5 nm for the FIB-SEM
and 10 Â 10 Â 10 nm for the SBEM, is quite sufficient for many of studies.
However, the FIB-SEM can produce 3D data at nanoscale resolutions sufficient to
visualise all organelles and large macromolecular complexes [10] and it is well
suited to smaller areas and achieving high resolutions. The SBEM on the other hand
can be an incredibly valuable tool when trying to gain structural data about a
biological system because it offers the opportunity to gain 3D data over a relatively
large area. With the SBEM an analysis of studies published have shown that typical
volumes can range from 5000–500,000 lm
3 and yet for the FIB-SEM it is only
20,000–100,000 lm
3 [18] highlighting the fact that the SBEM is a tool for capturing large volumes relative to the FIB-SEM.
5 Volume Scanning Electron Microscopy: Serial Block-Face …
141
needs to be monitored but with as minimal disturbance to the microscope as possible. If the dwell time necessary to achieve uniform milling is too low to achieve a
good SNR, reduce the FOV or increase the pixel size. It may also be necessary to
increase the FIB beam current or adjust the milling time by correcting the milling
rates and/or increasing the milling depth. Image acquisition and milling times
should be defined in relation to specimen drift [34]. There is always a balance
between these factors since the series of SEM images is collected automatically. If
the acquisition time is too long, the sample may either drift into the FIB beam thus
increasing the slice thickness or drift away from the FIB beam so that the surface is
burned by the imaging SEM beam and not milled away. As Maco et al. [75]
instructs, “any interruption to the imaging procedure will cause irregular thicknesses to be removed by the milling beam”. When parameters that produce quality
data with uniform milling are found it is crucial to minimise disturbances, with only
brief checks of focus and astigmatism [75].
5.5 Discussion
Volume SEM utilising the SBEM and FIB-SEM enables better appreciation of the
complexity of biological systems, which by nature are in three dimensions. They
produce a series of 2D images that are combined to create a 3D volume at the
nanoscale. This new generation of techniques has taken serial sectioning into a new
realm. It is now possible to understand the relative relationship between different
structures in a full volume rather than as individual two-dimensional images that are
obtained from routine TEM of ultrathin sections. Despite the existence of ssTEM
for more than 60 years, this method is limited by its laborious nature. With the
availability of commercial instruments that allow for automation, SBEM and
FIB-SEM have received wide acceptance in the scientific community and have now
made volume EM a routine technique.
While SBEM and FIB-SEM lack the ultimate resolution of a TEM, they fill a
major gap in resolution between light microscopy and the high end TEM. Many
biological studies do not require the highest resolution in order to answer the
scientific question being asked. A resolution of 5 Â 5 Â 5 nm for the FIB-SEM
and 10 Â 10 Â 10 nm for the SBEM, is quite sufficient for many of studies.
However, the FIB-SEM can produce 3D data at nanoscale resolutions sufficient to
visualise all organelles and large macromolecular complexes [10] and it is well
suited to smaller areas and achieving high resolutions. The SBEM on the other hand
can be an incredibly valuable tool when trying to gain structural data about a
biological system because it offers the opportunity to gain 3D data over a relatively
large area. With the SBEM an analysis of studies published have shown that typical
volumes can range from 5000–500,000 lm
3 and yet for the FIB-SEM it is only
20,000–100,000 lm
3 [18] highlighting the fact that the SBEM is a tool for capturing large volumes relative to the FIB-SEM.
5 Volume Scanning Electron Microscopy: Serial Block-Face …
141