After polymerisation the coverslips and plastic are removed to reveal a flat resin
disc containing the cell monolayer and imprinted co-ordinate pattern on one surface.
The outer plastic walls of the dish are carefully removed by a hacksaw with the dish
held in a clamp. The dish needs to be supported while sawing to end up with a
complete, unbroken disc. After the outer plastic walls are removed, the resin disc and
parts of the petri dish remain. This is then moved between liquid nitrogen and warm
water several times until the glass starts to separate from the resin. At this point a
thumb is placed over the central part where the resin disc is and pressure is applied.
The disc is broken out with the other hand. The glass should separate from both sides
so that all that remains is the resin disc that can be mounted on an SEM stub.
5.4.1.2 Microwave Processing
Here again the MatTek dish is used to process cell monolayers, however in this
method, all steps are carried out right through to polymerisation in a Pelco Biowave
Microwave (Ted Pella, Inc.) [69].
1. Primary fixation with 2x concentration in equal proportions to the cell media—
final concentration is 2.5% glutaraldehyde 0.05% malachite green oxalate in
0.1 M PHEM buffer left 1 min
2. Immediately followed by same 1x fixative in microwave for 7 alternating cycles
of 2 min at 100 W with vacuum
3. Two quick buffer bench rinses
4. Two 40 s buffer rinses in the microwave at 250 W without vacuum
5. 1% osmium tetroxide and 0.8% potassium ferricyanide in water 7 alternating
cycles of 2 min at 100 W with vacuum
6. Two quick water bench rinses
7. Two 40 s buffer rinses in the microwave at 250 W without vacuum
8. Aqueous 1% tannic acid 7 alternating cycles of 1 min at 150 W under vacuum
9. Two quick water bench rinses
10. Two 40 s buffer rinses in the microwave at 250 W without vacuum
11. 0.5% uranyl acetate in water 7 alternating cycles of 1 min at 150 W under
vacuum
12. Dehydration in a series of ethanol solutions each for 40 s at 250 W without
vacuum
13. Resin infiltration with Durcupan in a 2 step graded series for 3 min at 250 W
under vacuum for each step
14. Repeat steps 11–13 from Sect. 5.4.1.1
5.4.1.3 High Pressure Freezing and Freeze Substitution
Cell monolayers can also be observed in the FIB-SEM that are grown on sapphire
discs, high pressure frozen (HPF) and freeze substituted (FS) as in Villinger et al.
[66]. Below is a slight adaptation from this papers FS protocol.
136
R. I. Webb and N. L. Schieber
disc containing the cell monolayer and imprinted co-ordinate pattern on one surface.
The outer plastic walls of the dish are carefully removed by a hacksaw with the dish
held in a clamp. The dish needs to be supported while sawing to end up with a
complete, unbroken disc. After the outer plastic walls are removed, the resin disc and
parts of the petri dish remain. This is then moved between liquid nitrogen and warm
water several times until the glass starts to separate from the resin. At this point a
thumb is placed over the central part where the resin disc is and pressure is applied.
The disc is broken out with the other hand. The glass should separate from both sides
so that all that remains is the resin disc that can be mounted on an SEM stub.
5.4.1.2 Microwave Processing
Here again the MatTek dish is used to process cell monolayers, however in this
method, all steps are carried out right through to polymerisation in a Pelco Biowave
Microwave (Ted Pella, Inc.) [69].
1. Primary fixation with 2x concentration in equal proportions to the cell media—
final concentration is 2.5% glutaraldehyde 0.05% malachite green oxalate in
0.1 M PHEM buffer left 1 min
2. Immediately followed by same 1x fixative in microwave for 7 alternating cycles
of 2 min at 100 W with vacuum
3. Two quick buffer bench rinses
4. Two 40 s buffer rinses in the microwave at 250 W without vacuum
5. 1% osmium tetroxide and 0.8% potassium ferricyanide in water 7 alternating
cycles of 2 min at 100 W with vacuum
6. Two quick water bench rinses
7. Two 40 s buffer rinses in the microwave at 250 W without vacuum
8. Aqueous 1% tannic acid 7 alternating cycles of 1 min at 150 W under vacuum
9. Two quick water bench rinses
10. Two 40 s buffer rinses in the microwave at 250 W without vacuum
11. 0.5% uranyl acetate in water 7 alternating cycles of 1 min at 150 W under
vacuum
12. Dehydration in a series of ethanol solutions each for 40 s at 250 W without
vacuum
13. Resin infiltration with Durcupan in a 2 step graded series for 3 min at 250 W
under vacuum for each step
14. Repeat steps 11–13 from Sect. 5.4.1.1
5.4.1.3 High Pressure Freezing and Freeze Substitution
Cell monolayers can also be observed in the FIB-SEM that are grown on sapphire
discs, high pressure frozen (HPF) and freeze substituted (FS) as in Villinger et al.
[66]. Below is a slight adaptation from this papers FS protocol.
136
R. I. Webb and N. L. Schieber