5.3 Serial Block-Face Scanning Electron Microscopy
(SBF-SEM or SBEM)
Serial Block-Face Scanning Electron Microscopy (SBEM) is essentially an ultramicrotome built inside an SEM, and works on the same principle of removing thin
sections of material from the surface of the resin embedded sample. The sample is
then moved forward by a small increment and another section can be removed.
Images of the block-face are acquired between each section. This technology is
capable of providing detailed 3D data but has a restricted resolution along the z-axis
and is limited by sample charging and can have artefacts from sectioning [28, 34].
Cutting sections of 50 nm is routine in SBEM and it is possible to cut thinner.
Russell et al. [9] demonstrated that they could cut at 10 nm sections with the right
sample. The 3View uses an oscillating diamond knife. Studer [48] and Hashimoto
et al. [20] show that the oscillation does cause less damage while sectioning and
could be useful particularly for very thin sections. During the sectioning process the
sections tend to stick together and mount up as a group on the knife edge. These can
then be cleaned from the knife edge using compressed air at the end of the process.
In SBEM beam damage is particularly an issue. This means that it is not possible
to work at high magnifications in SBEM as when doing so the electron beam dose
is high in a small region of the sample. As a result, most SBEM work is done at
relatively low magnifications (generally below x10K). However, in an SEM it is
easy to change the pixel density of the image to gain large amounts of information
by enlarging the images taken at low magnifications. The Gatan 3View system
allows acquisition of images at up to 32k  24k and with the Maps software
available in the FEI VolumeScope it is possible to collect images at 40k  40k
pixels.
5.3.1 Sample Processing
SBEM is highly dependent on the processing of the sample. For a dedicated SBEM
project the samples should be processed with protocols using large amounts of
heavy metals as discussed below.
5.3.1.1 Conventional Bench Processing
A protocol designed in the NCMIR laboratory in San Diego has now become
widely used in the processing of samples for SBEM and is used with some slight
modifications in most laboratories [41, 42]. This standard protocol has an initial
osmium tetroxide step containing potassium ferrocyanide, which is known to give
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R. I. Webb and N. L. Schieber
(SBF-SEM or SBEM)
Serial Block-Face Scanning Electron Microscopy (SBEM) is essentially an ultramicrotome built inside an SEM, and works on the same principle of removing thin
sections of material from the surface of the resin embedded sample. The sample is
then moved forward by a small increment and another section can be removed.
Images of the block-face are acquired between each section. This technology is
capable of providing detailed 3D data but has a restricted resolution along the z-axis
and is limited by sample charging and can have artefacts from sectioning [28, 34].
Cutting sections of 50 nm is routine in SBEM and it is possible to cut thinner.
Russell et al. [9] demonstrated that they could cut at 10 nm sections with the right
sample. The 3View uses an oscillating diamond knife. Studer [48] and Hashimoto
et al. [20] show that the oscillation does cause less damage while sectioning and
could be useful particularly for very thin sections. During the sectioning process the
sections tend to stick together and mount up as a group on the knife edge. These can
then be cleaned from the knife edge using compressed air at the end of the process.
In SBEM beam damage is particularly an issue. This means that it is not possible
to work at high magnifications in SBEM as when doing so the electron beam dose
is high in a small region of the sample. As a result, most SBEM work is done at
relatively low magnifications (generally below x10K). However, in an SEM it is
easy to change the pixel density of the image to gain large amounts of information
by enlarging the images taken at low magnifications. The Gatan 3View system
allows acquisition of images at up to 32k  24k and with the Maps software
available in the FEI VolumeScope it is possible to collect images at 40k  40k
pixels.
5.3.1 Sample Processing
SBEM is highly dependent on the processing of the sample. For a dedicated SBEM
project the samples should be processed with protocols using large amounts of
heavy metals as discussed below.
5.3.1.1 Conventional Bench Processing
A protocol designed in the NCMIR laboratory in San Diego has now become
widely used in the processing of samples for SBEM and is used with some slight
modifications in most laboratories [41, 42]. This standard protocol has an initial
osmium tetroxide step containing potassium ferrocyanide, which is known to give
126
R. I. Webb and N. L. Schieber