can be fully processed from frozen sample to fully polymerised block in under one
working day.
1. Quick freeze substitution process in 1% imidazole 1% osmium tetroxide 1.5–
3 h, warm to room temperature
2. Wash in acetone, 3 Â 5 min
3. Thiocarbohydrazide 30 min
4. Wash in acetone, 3 Â 5 min
5. 2% osmium tetroxide 30 min
6. Wash in acetone, 3 Â 5 min
7. 2% uranyl acetate in acetone 30 min
8. Wash in acetone, 3 Â 5 min
9. 5% lead acetate in methanol 30 min
10. Wash in acetone, 3 Â 5 min
11. Infiltrate with Durcupan resin using microcentrifuge
12. Polymerise for 2 h at 90 °C
5.3.2 Sample Setup and Mounting
To prepare the embedded sample for the SBEM all excess resin must be removed to
the minimum amount possible without losing information. If the sample is stuck
down to the adherent surface of sticky tape it is relatively easy to trim with a razor
blade without losing the sample. Exposing the sample on all sides of the block is
important where possible, especially on the back side of the sample, so when it is
glued to the stub the sample itself is in contact with the metal. For cell cultures
embedded on a culture dish it is necessary to peel the polymerised resin off the dish
and cut out a very small region which is then trimmed as close as possible to the
cells. These samples can be mounted in two orientations on the stub, to achieve
either longitudinal or transverse sections. For CLEM projects it is important to
consider specific sample set ups for SBEM. Russell et al. [9] describe this process in
great detail for its application to cell monolayers that are grown in a dish with a
photo-etched grid pattern, making it possible to overlay 3D light microscopy onto
the 3D electron microscopy.
The sample stubs used in the 3View system, called rivets, are small pins with two
different head sizes (Fig. 5.2c). It is also possible to make rivets easily from jeweller’s
aluminium wire of 12 g (personal communication Graham Kidd) (Fig. 5.2d, e). A roll
of this wire can produce large numbers of rivets and costs very little. For the
VolumeScope it is necessary to use specific sample stubs that consist of a shaft with an
8 mm head. In the middle of the head is a 2 mm raised section for attachment of the
sample (Fig. 5.2c).
The sample is glued to the sample stub using a 2 part conductive epoxy glue
(CircuitWorks Conductive Epoxy CW2400). Placing it in a 60 °C oven can aid in
the setting of the glue. The sample is positioned according to the orientation needed
5 Volume Scanning Electron Microscopy: Serial Block-Face …
129
working day.
1. Quick freeze substitution process in 1% imidazole 1% osmium tetroxide 1.5–
3 h, warm to room temperature
2. Wash in acetone, 3 Â 5 min
3. Thiocarbohydrazide 30 min
4. Wash in acetone, 3 Â 5 min
5. 2% osmium tetroxide 30 min
6. Wash in acetone, 3 Â 5 min
7. 2% uranyl acetate in acetone 30 min
8. Wash in acetone, 3 Â 5 min
9. 5% lead acetate in methanol 30 min
10. Wash in acetone, 3 Â 5 min
11. Infiltrate with Durcupan resin using microcentrifuge
12. Polymerise for 2 h at 90 °C
5.3.2 Sample Setup and Mounting
To prepare the embedded sample for the SBEM all excess resin must be removed to
the minimum amount possible without losing information. If the sample is stuck
down to the adherent surface of sticky tape it is relatively easy to trim with a razor
blade without losing the sample. Exposing the sample on all sides of the block is
important where possible, especially on the back side of the sample, so when it is
glued to the stub the sample itself is in contact with the metal. For cell cultures
embedded on a culture dish it is necessary to peel the polymerised resin off the dish
and cut out a very small region which is then trimmed as close as possible to the
cells. These samples can be mounted in two orientations on the stub, to achieve
either longitudinal or transverse sections. For CLEM projects it is important to
consider specific sample set ups for SBEM. Russell et al. [9] describe this process in
great detail for its application to cell monolayers that are grown in a dish with a
photo-etched grid pattern, making it possible to overlay 3D light microscopy onto
the 3D electron microscopy.
The sample stubs used in the 3View system, called rivets, are small pins with two
different head sizes (Fig. 5.2c). It is also possible to make rivets easily from jeweller’s
aluminium wire of 12 g (personal communication Graham Kidd) (Fig. 5.2d, e). A roll
of this wire can produce large numbers of rivets and costs very little. For the
VolumeScope it is necessary to use specific sample stubs that consist of a shaft with an
8 mm head. In the middle of the head is a 2 mm raised section for attachment of the
sample (Fig. 5.2c).
The sample is glued to the sample stub using a 2 part conductive epoxy glue
(CircuitWorks Conductive Epoxy CW2400). Placing it in a 60 °C oven can aid in
the setting of the glue. The sample is positioned according to the orientation needed
5 Volume Scanning Electron Microscopy: Serial Block-Face …
129