For many years the only way to gain 3D data on biological systems was to use
serial section transmission electron microscopy (ssTEM). While this technique has
been used almost since ultramicrotomy was invented [1, 2], it has never been widely
accepted because of the technical challenges. It requires cutting large numbers of
serial sections that are transferred to a grid and taken to the transmission electron
microscope (TEM) as a full intact series. The individual imaging of each section and
the consequent realigning is also a bottleneck since it is generally done manually.
The most complete example of ssTEM was performed on Caenorhabditis elegans
where the entire nervous system was reconstructed [3].
Technological advances have moved the field beyond ssTEM in terms of
throughput and automation with volume scanning electron microscopy (SEM). Two
forms of this are microtome based Serial Block-Face SEM (SBEM, also named
SBF-SEM in some reports), and Focused Ion Beam SEM (FIB-SEM). These
methods both sequentially image the sample surface by SEM after the removal of
material by either cutting or milling. They have sped up and automated a process,
which previously required skilled personnel plus a lot of slow, tedious work
(ssTEM).
In this chapter we will focus on two versions of volume-SEM, SBEM and
FIB-SEM. The specific sample preparation is a fundamental part of these techniques, so here our aim is to provide a more practical approach to both preparing
biological samples for such microscopes and to discuss the operation of the
systems.
5.2 Volume Scanning Electron Microscopy—SBEM
and FIB-SEM
SBEM and FIB-SEM technologies have successfully opened up possibilities to
visualize biological samples in three dimensions [4–9]. From resin embedded
biological material the SBEM, uses an integrated ultramicrotome to cut away
material and FIB-SEM, uses a gallium focused ion beam to mill it away (Fig. 5.1
schematic). In an iterative process, the SEM then images the newly generated
surface and the collection of these 2D images can be converted to a 3D ultrastructural volume. Both techniques are destructive, so it is important to consider
exactly what is wanted from a SBEM or FIB-SEM project before it begins.
FIB-SEM and SBEM give similar results but each has their limitations. Titze and
Genoud [10] and Briggman and Bock [11] give comprehensive overviews of these
3D EM techniques, as well as others.
Volume SEM gives access to similar information to that typically acquired in a
TEM. Indeed, they both rely on the interaction of the electron beam with heavy
metal stained cellular components. However, the lateral resolution of TEM is higher
than in SEM (due to many factors including the higher acceleration voltage in
TEM and the probe size in SEM). Furthermore, TEM is a wide-field technique,
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serial section transmission electron microscopy (ssTEM). While this technique has
been used almost since ultramicrotomy was invented [1, 2], it has never been widely
accepted because of the technical challenges. It requires cutting large numbers of
serial sections that are transferred to a grid and taken to the transmission electron
microscope (TEM) as a full intact series. The individual imaging of each section and
the consequent realigning is also a bottleneck since it is generally done manually.
The most complete example of ssTEM was performed on Caenorhabditis elegans
where the entire nervous system was reconstructed [3].
Technological advances have moved the field beyond ssTEM in terms of
throughput and automation with volume scanning electron microscopy (SEM). Two
forms of this are microtome based Serial Block-Face SEM (SBEM, also named
SBF-SEM in some reports), and Focused Ion Beam SEM (FIB-SEM). These
methods both sequentially image the sample surface by SEM after the removal of
material by either cutting or milling. They have sped up and automated a process,
which previously required skilled personnel plus a lot of slow, tedious work
(ssTEM).
In this chapter we will focus on two versions of volume-SEM, SBEM and
FIB-SEM. The specific sample preparation is a fundamental part of these techniques, so here our aim is to provide a more practical approach to both preparing
biological samples for such microscopes and to discuss the operation of the
systems.
5.2 Volume Scanning Electron Microscopy—SBEM
and FIB-SEM
SBEM and FIB-SEM technologies have successfully opened up possibilities to
visualize biological samples in three dimensions [4–9]. From resin embedded
biological material the SBEM, uses an integrated ultramicrotome to cut away
material and FIB-SEM, uses a gallium focused ion beam to mill it away (Fig. 5.1
schematic). In an iterative process, the SEM then images the newly generated
surface and the collection of these 2D images can be converted to a 3D ultrastructural volume. Both techniques are destructive, so it is important to consider
exactly what is wanted from a SBEM or FIB-SEM project before it begins.
FIB-SEM and SBEM give similar results but each has their limitations. Titze and
Genoud [10] and Briggman and Bock [11] give comprehensive overviews of these
3D EM techniques, as well as others.
Volume SEM gives access to similar information to that typically acquired in a
TEM. Indeed, they both rely on the interaction of the electron beam with heavy
metal stained cellular components. However, the lateral resolution of TEM is higher
than in SEM (due to many factors including the higher acceleration voltage in
TEM and the probe size in SEM). Furthermore, TEM is a wide-field technique,
118
R. I. Webb and N. L. Schieber