5.3.1.2 Microwave Processing
The sample protocol listed above is quite long lasting 5 days. To speed up the
process and aid with the diffusion of chemicals the Pelco Biowave microwave (Ted
Pella Inc.) can also be used for all electron microscopy processing [44]. A protocol
utilising a Ted Pella Biowave processing microwave oven is given below.
1. 2% osmium tetroxide 1.5% potassium ferricyanide 4 cycles of 2 min at 80 W
with vacuum
2. Quick bench water wash, then 2 Â 40 s 80 W with vacuum
3. 1% thiocarbohydrazide 6 alternating cycles of 2 min at 80 W with vacuum
4. Quick bench water wash, then 2 Â 40 s 80 W with vacuum 2% osmium
tetroxide 6 alternating cycles of 2 min at 80 W with vacuum
5. Quick bench water wash, then 2 Â 40 s 80 W with vacuum
6. 1% uranyl acetate (aqueous) 6 alternating cycles of 2 min at 80 W with vacuum
7. Quick bench water wash, then 2 Â 40 s 80 W with vacuum
8. Lead aspartate at 50 °C 6 alternating cycles of 2 min at 80 W with vacuum
9. Quick bench water wash, then 2 Â 40 s 80 W with vacuum
10. Dehydration and infiltration at 250 W with vacuum
11. Polymerisation at 60 °C in a normal oven for 2 days.
For the lead staining the temperature of the Steady Temp is turned up to 50 °C
and the sample processing vials or cell culture dishes are placed directly onto the
Cold Spot inside the chamber of the Biowave. This will allow warming of the
samples during the en bloc staining with Walton’s lead aspartate. Kremer et al. [16]
also list a slightly different microwave protocol for SBEM sample processing.
5.3.1.3 High Pressure Freezing and Freeze Substitution
High pressure freezing and freeze substitution give superior structural preservation
to standard room temperature processing [40]. Standard freeze substitution protocols do not impregnate sufficient metals into the sample to allow their use with
SBEM. Webb and Webb [54] have introduced an extended freeze substitution
protocol that is suitable for SBEM. Below is their protocol based on the quick
freeze substitution protocol of McDonald and Webb [55]. Thiocarbohydrazide is
not particularly soluble in acetone but enough becomes available in solution to be
used for this work. It is dissolved at 0.05 g in 5 ml of acetone. After mixing
thoroughly and heating at 60 °C for an hour this stock solution is diluted 1:10 with
acetone (personal communication Shigeki Watanabe). Lead acetate at 5% is made
by dissolving it in methanol [56]. Once the freeze substitution has reached room
temperature and the samples are washed in acetone, the remainder of the process is
performed at room temperature. Agitation for all steps is important in enhancing
diffusion of the solutions and also helps with the acetone washes. When this
technique is combined with quick processing following McDonald [40] samples
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The sample protocol listed above is quite long lasting 5 days. To speed up the
process and aid with the diffusion of chemicals the Pelco Biowave microwave (Ted
Pella Inc.) can also be used for all electron microscopy processing [44]. A protocol
utilising a Ted Pella Biowave processing microwave oven is given below.
1. 2% osmium tetroxide 1.5% potassium ferricyanide 4 cycles of 2 min at 80 W
with vacuum
2. Quick bench water wash, then 2 Â 40 s 80 W with vacuum
3. 1% thiocarbohydrazide 6 alternating cycles of 2 min at 80 W with vacuum
4. Quick bench water wash, then 2 Â 40 s 80 W with vacuum 2% osmium
tetroxide 6 alternating cycles of 2 min at 80 W with vacuum
5. Quick bench water wash, then 2 Â 40 s 80 W with vacuum
6. 1% uranyl acetate (aqueous) 6 alternating cycles of 2 min at 80 W with vacuum
7. Quick bench water wash, then 2 Â 40 s 80 W with vacuum
8. Lead aspartate at 50 °C 6 alternating cycles of 2 min at 80 W with vacuum
9. Quick bench water wash, then 2 Â 40 s 80 W with vacuum
10. Dehydration and infiltration at 250 W with vacuum
11. Polymerisation at 60 °C in a normal oven for 2 days.
For the lead staining the temperature of the Steady Temp is turned up to 50 °C
and the sample processing vials or cell culture dishes are placed directly onto the
Cold Spot inside the chamber of the Biowave. This will allow warming of the
samples during the en bloc staining with Walton’s lead aspartate. Kremer et al. [16]
also list a slightly different microwave protocol for SBEM sample processing.
5.3.1.3 High Pressure Freezing and Freeze Substitution
High pressure freezing and freeze substitution give superior structural preservation
to standard room temperature processing [40]. Standard freeze substitution protocols do not impregnate sufficient metals into the sample to allow their use with
SBEM. Webb and Webb [54] have introduced an extended freeze substitution
protocol that is suitable for SBEM. Below is their protocol based on the quick
freeze substitution protocol of McDonald and Webb [55]. Thiocarbohydrazide is
not particularly soluble in acetone but enough becomes available in solution to be
used for this work. It is dissolved at 0.05 g in 5 ml of acetone. After mixing
thoroughly and heating at 60 °C for an hour this stock solution is diluted 1:10 with
acetone (personal communication Shigeki Watanabe). Lead acetate at 5% is made
by dissolving it in methanol [56]. Once the freeze substitution has reached room
temperature and the samples are washed in acetone, the remainder of the process is
performed at room temperature. Agitation for all steps is important in enhancing
diffusion of the solutions and also helps with the acetone washes. When this
technique is combined with quick processing following McDonald [40] samples
128
R. I. Webb and N. L. Schieber