1. Substituted in 1% osmium tetroxide 0.5% uranyl acetate 5% water in acetone
2. −90–20 °C with a 5 °C/hour slope
3. Kept at 20 °C for up to one hour
4. 3 Â 40 s acetone rinses at 250 W without vacuum in the microwave
5. Resin infiltration with Durcupan in a two step graded series for 3 minutes at
250 W under vacuum for each step
6. Processed sapphire discs are place into an eppendorf tube, see Villinger et al. [66]
7. Polymerized at 60 °C for 3 days.
5.4.2 Sample Setup and Mounting
Before embarking on FIB-SEM, check the quality of samples via routine TEM
inspection to look for good preservation and sufficient sample contrast. When
imaging monolayers prepared as described above (both chemically fixed and HPF),
the cells are directly exposed to FIB-SEM imaging after removal of the coverslip
(or sapphire disc). They are mounted cell side up onto an SEM stub (for illustration
of chemically fixed cell sample see Fig. 5.6g–i) with carbon conductive cement
(Plano) or 2 part conductive epoxy glue (CircuitWorks Conductive Epoxy
CW2400). The mounting of HPF cell samples is described by Villinger et al. [66].
The region of interest needs to be positioned as close as possible to the upper
edge of the block surface in relation to the FIB beam (surface 1 in Fig. 5.6e). The
top of the cross section will have the best imaging surface since it is where the ion
beam is most focused. The focus of the beam is decreased the deeper it penetrates,
therefore reducing the milling effect and decreasing the chance that ions will be able
to sputter molecules away from the block [7].
For tissues, it is necessary to trim the sample. Strategies for exposing specific
targets within bulk specimens have been developed and described previously (see
Karreman et al. [37] and Maco et al. [75]). If this is not required, they need to be
trimmed to expose the sample surface at two faces at the microtome (Fig. 5.6a–f).
One of these surfaces will become the imaging surface (surface 2 in Fig. 5.6e), the
other will be perpendicular to the FIB and the deposition will be placed here over
your ROI (surface 1 in Fig. 5.6e). Processed blocks are first trimmed with a razor
blade and then with a 90° diamond knife to get smooth surfaces (Fig. 5.6a). This
can then be removed from the block with a sharp razor blade using double sided or
masking tape to stop the piece being lost (Fig. 5.6b). The very small block will
remain attached to the double sided tape (Fig. 5.6c) and can be trimmed to give a
flat surface to mount directly onto the SEM stub (Fig. 5.6d, white arrow head).
For all samples, once mounted on an SEM stub with a conductive cement or
glue, each are carefully surrounded with silver paint (Colloidal Silver Liquid, Ted
Pella, Inc.), so as to not cover any regions of interest but to ensure it is well
grounded to the specimen stub (Fig. 5.6d, g). Then the samples are sputter coated
with a thin layer of gold, or carbon. These applications help to dampen charge
accumulation in the sample [34].
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137
2. −90–20 °C with a 5 °C/hour slope
3. Kept at 20 °C for up to one hour
4. 3 Â 40 s acetone rinses at 250 W without vacuum in the microwave
5. Resin infiltration with Durcupan in a two step graded series for 3 minutes at
250 W under vacuum for each step
6. Processed sapphire discs are place into an eppendorf tube, see Villinger et al. [66]
7. Polymerized at 60 °C for 3 days.
5.4.2 Sample Setup and Mounting
Before embarking on FIB-SEM, check the quality of samples via routine TEM
inspection to look for good preservation and sufficient sample contrast. When
imaging monolayers prepared as described above (both chemically fixed and HPF),
the cells are directly exposed to FIB-SEM imaging after removal of the coverslip
(or sapphire disc). They are mounted cell side up onto an SEM stub (for illustration
of chemically fixed cell sample see Fig. 5.6g–i) with carbon conductive cement
(Plano) or 2 part conductive epoxy glue (CircuitWorks Conductive Epoxy
CW2400). The mounting of HPF cell samples is described by Villinger et al. [66].
The region of interest needs to be positioned as close as possible to the upper
edge of the block surface in relation to the FIB beam (surface 1 in Fig. 5.6e). The
top of the cross section will have the best imaging surface since it is where the ion
beam is most focused. The focus of the beam is decreased the deeper it penetrates,
therefore reducing the milling effect and decreasing the chance that ions will be able
to sputter molecules away from the block [7].
For tissues, it is necessary to trim the sample. Strategies for exposing specific
targets within bulk specimens have been developed and described previously (see
Karreman et al. [37] and Maco et al. [75]). If this is not required, they need to be
trimmed to expose the sample surface at two faces at the microtome (Fig. 5.6a–f).
One of these surfaces will become the imaging surface (surface 2 in Fig. 5.6e), the
other will be perpendicular to the FIB and the deposition will be placed here over
your ROI (surface 1 in Fig. 5.6e). Processed blocks are first trimmed with a razor
blade and then with a 90° diamond knife to get smooth surfaces (Fig. 5.6a). This
can then be removed from the block with a sharp razor blade using double sided or
masking tape to stop the piece being lost (Fig. 5.6b). The very small block will
remain attached to the double sided tape (Fig. 5.6c) and can be trimmed to give a
flat surface to mount directly onto the SEM stub (Fig. 5.6d, white arrow head).
For all samples, once mounted on an SEM stub with a conductive cement or
glue, each are carefully surrounded with silver paint (Colloidal Silver Liquid, Ted
Pella, Inc.), so as to not cover any regions of interest but to ensure it is well
grounded to the specimen stub (Fig. 5.6d, g). Then the samples are sputter coated
with a thin layer of gold, or carbon. These applications help to dampen charge
accumulation in the sample [34].
5 Volume Scanning Electron Microscopy: Serial Block-Face …
137