It is a lot of trial and error in setting up a SBEM to acquire a dataset. For similar
samples it is quite often possible to use the same settings thus simplifying the
process. As a guide this is always a good place to start and then to adjust the system
as necessary. Once it appears that the sectioning and imaging will be stable the run
can be commenced. However, it good to keep checking the SBEM during the run to
ensure that it is still working well. All of this testing on a sample can mean that a
reasonable amount of material is removed before the run is started, so it’s important
to take this into consideration when setting up the sample.
5.4 Focused Ion Beam Scanning Electron Microscopy
Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) was also previously
known as Ion Abrasion Scanning Electron Microscopy (IA-SEM) [64]. It uses a
focused ion beam (typically gallium, Ga
þ ) to mill away slices from
resin-embedded (or cryo-fixed) samples and then images the newly generated
surface by the SEM in an iterative process in order to build a 3D dataset [6, 65].
The FIB operates similar to the SEM whereby a beam of charged particles is
rastered across a specimen, and the resultant signals at each raster position are
plotted to form an image. However, it uses ions instead of electrons to form the
beam. The ions are massive relative to electrons, and they interact with the sample
and cause sputtering. At low beam currents, the FIB gallium ions are used for
imaging while high beam currents are used for site-specific sputtering [29].
The FIB coupled with an SEM, has proven to be a versatile and powerful tool in
material and biological research alike. A basic FIB-SEM instrument consists of a
LMIS, an ion column, a sample stage, an electron column and gas inlets.
Commercial instruments have an SEM column that is usually oriented vertically
and a FIB column that is oriented in the range of 45°–55° relative to the electron
column. Both beams can access a sample in the FIB-SEM chamber simultaneously
when they are at the coincidence point. 3D acquisitions are normally done with the
sample tilted so that its surface is orthogonal to the FIB and the SEM is viewing the
sample at an angle (Fig. 5.1). Section 5.4.3 describes an example of this method of
FIB-SEM operation drawing predominately from the Zeiss systems. This
arrangement is preferred as it is efficient and stable with only minor compromises to
the resolution and signal [6].
The ion and electron beams complement each other in charge reduction, protective depositions and imaging information. The electron beam can be used to
monitor the ion beam milling to target precisely the feature of interest, while
allowing non-destructive imaging of the sample. FIB-SEM allows sample preparation, imaging and analysis to be accomplished in one tool. It is used to obtain
isotropic datasets from biological samples so that the resolution in z is equal to the
lateral pixel resolution in xy. Reports have been made of consistent slice thickness
around 3 nm, with the Atlas 3D software [34]. More routine collection over FOVs
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R. I. Webb and N. L. Schieber
samples it is quite often possible to use the same settings thus simplifying the
process. As a guide this is always a good place to start and then to adjust the system
as necessary. Once it appears that the sectioning and imaging will be stable the run
can be commenced. However, it good to keep checking the SBEM during the run to
ensure that it is still working well. All of this testing on a sample can mean that a
reasonable amount of material is removed before the run is started, so it’s important
to take this into consideration when setting up the sample.
5.4 Focused Ion Beam Scanning Electron Microscopy
Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) was also previously
known as Ion Abrasion Scanning Electron Microscopy (IA-SEM) [64]. It uses a
focused ion beam (typically gallium, Ga
þ ) to mill away slices from
resin-embedded (or cryo-fixed) samples and then images the newly generated
surface by the SEM in an iterative process in order to build a 3D dataset [6, 65].
The FIB operates similar to the SEM whereby a beam of charged particles is
rastered across a specimen, and the resultant signals at each raster position are
plotted to form an image. However, it uses ions instead of electrons to form the
beam. The ions are massive relative to electrons, and they interact with the sample
and cause sputtering. At low beam currents, the FIB gallium ions are used for
imaging while high beam currents are used for site-specific sputtering [29].
The FIB coupled with an SEM, has proven to be a versatile and powerful tool in
material and biological research alike. A basic FIB-SEM instrument consists of a
LMIS, an ion column, a sample stage, an electron column and gas inlets.
Commercial instruments have an SEM column that is usually oriented vertically
and a FIB column that is oriented in the range of 45°–55° relative to the electron
column. Both beams can access a sample in the FIB-SEM chamber simultaneously
when they are at the coincidence point. 3D acquisitions are normally done with the
sample tilted so that its surface is orthogonal to the FIB and the SEM is viewing the
sample at an angle (Fig. 5.1). Section 5.4.3 describes an example of this method of
FIB-SEM operation drawing predominately from the Zeiss systems. This
arrangement is preferred as it is efficient and stable with only minor compromises to
the resolution and signal [6].
The ion and electron beams complement each other in charge reduction, protective depositions and imaging information. The electron beam can be used to
monitor the ion beam milling to target precisely the feature of interest, while
allowing non-destructive imaging of the sample. FIB-SEM allows sample preparation, imaging and analysis to be accomplished in one tool. It is used to obtain
isotropic datasets from biological samples so that the resolution in z is equal to the
lateral pixel resolution in xy. Reports have been made of consistent slice thickness
around 3 nm, with the Atlas 3D software [34]. More routine collection over FOVs
134
R. I. Webb and N. L. Schieber