for sectioning in the SBEM. If the sample already has a smoothened surface ready
for sectioning (for example, cultured cells grown on a dish) it is important to try to
make the block-face almost but not quite perpendicular to the shaft of the mount. In
this way it will be almost parallel to the cutting action of the diamond knife in the
SBEM.
Further trimming of the sample can be performed on an ultramicrotome stage.
Using the trimming adapter (Fig. 5.2f) or the segment arc set on 0° is useful so that
the sample will be aligned for the SBEM. The cutting window in the 3View is only
1.2 mm and 2 mm in the VolumeScope, so the block-face should be smaller than
this window. Semi-thin or even ultrathin sections can be taken from the block to
find the ROI and to check the quality of preservation of the sample.
Conductive silver paint (Colloidal Silver Liquid, Ted Pella, Inc.) is then put
around the sample on all sides making a layer that is continuous around the entire
sample and that also connects it to the sample stub (Fig. 5.2d, e). Placing the
sample in a 60 °C oven will completely dry this paint. Finally, the mounted sample
is sputter coated with a thin layer of platinum to make a continuous coat over the
entire surface. Altogether, the conductive epoxy glue, the silver paint and the
sputter coating will give conductivity to the sample in an attempt to reduce
charging.
Samples ready for SBEM can be stored inside the chamber so they are kept
under vacuum. This helps to outgas the resin and stabilize the samples. The sample
is inserted into the stage of the SBEM and aligned with the SEM door opened. This
alignment involves setting up the sample so it is in the correct position relative to
the diamond knife. As in an ultramicrotome, the sample positioning is done
watching reflections that are seen on the sample block-face. The reflections
(Fig. 5.2g) are used to judge the distance between the sample block-face and the
diamond knife and the sample is raised until this reflection almost vanishes so that
the sample and knife are almost touching. For most samples the initial sectioning is
still part of the alignment and is done while the door is still open. The sectioning
can be performed at a greater section thickness (200 nm) and a faster cutting speed
(1.2 mm/sec). Once the entire block-face has been sectioned the process is stopped,
the sections are removed from the knife edge with compressed air (Fig. 5.2h), the
door of the SEM is closed and the chamber is pumped down. For cultured cells
being sectioned longitudinally it is important to stop this coarse approach as soon as
the first sections come off.
After the chamber has reached vacuum wait for the sample to stabilise and
outgas fully. At least an hour is advisable.
5.3.3 SBEM Operation
Once the SEM is pumped down and the sample is stable the electron beam can be
turned on and the sample imaged. Some prior knowledge of the system and its
operation on similar samples will give a starting point (for example, the voltage or
130
R. I. Webb and N. L. Schieber
for sectioning (for example, cultured cells grown on a dish) it is important to try to
make the block-face almost but not quite perpendicular to the shaft of the mount. In
this way it will be almost parallel to the cutting action of the diamond knife in the
SBEM.
Further trimming of the sample can be performed on an ultramicrotome stage.
Using the trimming adapter (Fig. 5.2f) or the segment arc set on 0° is useful so that
the sample will be aligned for the SBEM. The cutting window in the 3View is only
1.2 mm and 2 mm in the VolumeScope, so the block-face should be smaller than
this window. Semi-thin or even ultrathin sections can be taken from the block to
find the ROI and to check the quality of preservation of the sample.
Conductive silver paint (Colloidal Silver Liquid, Ted Pella, Inc.) is then put
around the sample on all sides making a layer that is continuous around the entire
sample and that also connects it to the sample stub (Fig. 5.2d, e). Placing the
sample in a 60 °C oven will completely dry this paint. Finally, the mounted sample
is sputter coated with a thin layer of platinum to make a continuous coat over the
entire surface. Altogether, the conductive epoxy glue, the silver paint and the
sputter coating will give conductivity to the sample in an attempt to reduce
charging.
Samples ready for SBEM can be stored inside the chamber so they are kept
under vacuum. This helps to outgas the resin and stabilize the samples. The sample
is inserted into the stage of the SBEM and aligned with the SEM door opened. This
alignment involves setting up the sample so it is in the correct position relative to
the diamond knife. As in an ultramicrotome, the sample positioning is done
watching reflections that are seen on the sample block-face. The reflections
(Fig. 5.2g) are used to judge the distance between the sample block-face and the
diamond knife and the sample is raised until this reflection almost vanishes so that
the sample and knife are almost touching. For most samples the initial sectioning is
still part of the alignment and is done while the door is still open. The sectioning
can be performed at a greater section thickness (200 nm) and a faster cutting speed
(1.2 mm/sec). Once the entire block-face has been sectioned the process is stopped,
the sections are removed from the knife edge with compressed air (Fig. 5.2h), the
door of the SEM is closed and the chamber is pumped down. For cultured cells
being sectioned longitudinally it is important to stop this coarse approach as soon as
the first sections come off.
After the chamber has reached vacuum wait for the sample to stabilise and
outgas fully. At least an hour is advisable.
5.3.3 SBEM Operation
Once the SEM is pumped down and the sample is stable the electron beam can be
turned on and the sample imaged. Some prior knowledge of the system and its
operation on similar samples will give a starting point (for example, the voltage or
130
R. I. Webb and N. L. Schieber