5.4.3 FIB-SEM Operation
For efficient use of the FIB-SEM, there are some considerations aside from proper
mounting that will give the best possible results. Make sure those of the gas
injection systems (GIS) that will be used are turned on with enough time to warm
up. This keeps them heated, stable and ready to use. Perform FIB alignments using
a Faraday cup and silicon mat to ensure the best possible beam profile. Give the
sample time to stabilise by loading them at least one day before starting the
acquisition so that it can out gas and after the final polish wait for at least 1 h to
allow the sample holder and sample to reach thermal equilibrium with the microscope stage and chamber [75]. The process of sample preparation should be documented very well since the sample will likely look very different in the SEM. If
possible, build a map either with CLEM or by overlaying different imaging
modalities to outline exactly where to image.
There are many papers explaining the intricacies of acquiring data with a
FIB-SEM [6, 7, 21, 32, 64, 65, 72, 75–80]. In general, the approach is similar for all
biological samples, although there are many things to optimise depending on your
requirements and the system you are operating. The following description of
microscope operation is based on the Zeiss systems with the aim to outline details
that can be utilised from any FIB-SEM instrument.
When the sample is in the FIB-SEM, before the column valves are opened,
outgas the GIS so that the lines are clear before use and to be sure there is no
contamination in the reservoir. Turn on the SEM and FIB. For SEM, an electron
high tension (EHT) of 5 kV is a good starting point for biological samples. HPF
samples may need higher values to see the material, which is deeper below the resin
surface. Position the specimen stub under the SEM beam and at low magnification
find the sample. Set the eucentric height by centring a feature on the specimen
surface when tilted to a few different angles between 0° and 54°. Set the working
distance and optimise SEM imaging conditions with crisp focus and astigmatism.
With a FIB imaging current of 50 pA make a good image. Even at low currents
such as this, the FIB will damage the sample surface so do not image unnecessarily.
Set the coincidence so that both the SEM and FIB beams are looking at the same
point.
Once the microscope is aligned to the sample, locate the region of interest and
begin the sample preparation. Regardless of the instrument in use, the first step is to
deposit a protective coat by ionising the precursor gas with the FIB (Fig. 5.6h, PC).
This coat will protect the surface of the sample and ensure even milling that is
parallel to the direction of the ion beam [34, 75]. This is particularly important if the
surface of the sample has some topography or when imaging surfaces of heterogeneous material composition [7]. Without it, there may be streaking or vertical
stripes down the block face, known as curtaining artefact [81]. After inserting the
GIS nozzle, note that it acts as an antenna and creates a small beam shift, check that
the assigned position of the deposition still corresponds to the targeted ROI. The
size of this deposition will depend on the ROI to be imaged. As a starting point,
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For efficient use of the FIB-SEM, there are some considerations aside from proper
mounting that will give the best possible results. Make sure those of the gas
injection systems (GIS) that will be used are turned on with enough time to warm
up. This keeps them heated, stable and ready to use. Perform FIB alignments using
a Faraday cup and silicon mat to ensure the best possible beam profile. Give the
sample time to stabilise by loading them at least one day before starting the
acquisition so that it can out gas and after the final polish wait for at least 1 h to
allow the sample holder and sample to reach thermal equilibrium with the microscope stage and chamber [75]. The process of sample preparation should be documented very well since the sample will likely look very different in the SEM. If
possible, build a map either with CLEM or by overlaying different imaging
modalities to outline exactly where to image.
There are many papers explaining the intricacies of acquiring data with a
FIB-SEM [6, 7, 21, 32, 64, 65, 72, 75–80]. In general, the approach is similar for all
biological samples, although there are many things to optimise depending on your
requirements and the system you are operating. The following description of
microscope operation is based on the Zeiss systems with the aim to outline details
that can be utilised from any FIB-SEM instrument.
When the sample is in the FIB-SEM, before the column valves are opened,
outgas the GIS so that the lines are clear before use and to be sure there is no
contamination in the reservoir. Turn on the SEM and FIB. For SEM, an electron
high tension (EHT) of 5 kV is a good starting point for biological samples. HPF
samples may need higher values to see the material, which is deeper below the resin
surface. Position the specimen stub under the SEM beam and at low magnification
find the sample. Set the eucentric height by centring a feature on the specimen
surface when tilted to a few different angles between 0° and 54°. Set the working
distance and optimise SEM imaging conditions with crisp focus and astigmatism.
With a FIB imaging current of 50 pA make a good image. Even at low currents
such as this, the FIB will damage the sample surface so do not image unnecessarily.
Set the coincidence so that both the SEM and FIB beams are looking at the same
point.
Once the microscope is aligned to the sample, locate the region of interest and
begin the sample preparation. Regardless of the instrument in use, the first step is to
deposit a protective coat by ionising the precursor gas with the FIB (Fig. 5.6h, PC).
This coat will protect the surface of the sample and ensure even milling that is
parallel to the direction of the ion beam [34, 75]. This is particularly important if the
surface of the sample has some topography or when imaging surfaces of heterogeneous material composition [7]. Without it, there may be streaking or vertical
stripes down the block face, known as curtaining artefact [81]. After inserting the
GIS nozzle, note that it acts as an antenna and creates a small beam shift, check that
the assigned position of the deposition still corresponds to the targeted ROI. The
size of this deposition will depend on the ROI to be imaged. As a starting point,
5 Volume Scanning Electron Microscopy: Serial Block-Face …
139