Leaves at this stage were approximately 3.5cm in length and 1.5cm in width. Samples were taken from the middle of the leaves close to the middle vein. Small sections of leaves (1mm²) were cut with a razor blade on a modelling wax plate in a drop of 3% glutaraldehyde in 0.06M Sørensen phosphate buffer at pH 7.2.
Sections were then evacuated with a water jet vacuum pump for a maximum of 10 seconds in a vial filled with the above described fixative solution. Subsequently, the specimens were transferred into small baskets with a mesh width of about 200μm. These baskets were then stacked on top of each other and transferred into the mono-mode chamber of the Leica EM AMW which already contained a vial filled with the above mentioned fixative solution. Microwave fixation was then started about 2 minutes after the cutting of the samples by starting the previously programmed protocol.
Sample preparation for transmission electron microscopy (TEM) was performed in order to develop a standard protocol that would reduce sample preparation time for TEM-investigations. Therefore the overall and fine structure of leaf cells prepared with the Leica EM AMW were compared with leaf cells that were prepared with a conventional fixation protocol at room temperature. Additionally, the diameter of membranes from different cell compartments (chloroplasts, nuclei and plasmamembrane) was determined by using quantitative computer supported transmission electron microscopy.
Epon, Agar 100
Standard mixture: Glauert Medium
| Epon (Agar 100 Harz, Fa. Gröpl) | 20ml | 24g |
|---|---|---|
| DDSA (Dodecenylsuccinic adhydrid) | 16ml | 16g |
| MNA (Methylnadicanhydrid) | 8ml | 10g |
| BDMA (Benzyldimethylamin) | 1,3ml | 1,2g |
| Epon soft: | Epon hard: | ||
|---|---|---|---|
| Epon | 24g | Epon | 24g |
| DDSA | 22g | DDSA | 9g |
| MNA | 6g | MNA | 15g |
| BDMA | 1,2g | BDMA | 1,2g |
| Processing | ||||||
| vial | step | time | max. temp. (°C) | reagent | mode | max. power (W) |
| 1 | 1 | 00:02:00 | 37 | Buffer + glutar aldehyde | Cont. | 15 |
| 1 | 2 | 00:02:00 | 20 | Buffer + glutar aldehyde | Cont. | 0 |
| 1 | 3 | 00:02:00 | 37 | Buffer + glutar aldehyde | Cont. | 15 |
| 1 | 4 | 00:02:00 | 20 | Buffer + glutar aldehyde | Cont. | 0 |
| 2 | 1 | 00:00:40 | 37 | Buffer | Slope | 20 |
| 3 | 1 | 00:00:40 | 37 | Buffer | Pulse | 15 |
| 4 | 1 | 00:00:40 | 37 | Buffer | Slope | 20 |
| 5 | 1 | 00:12:00 | 37 | Buffer + OsO4 | Cont. | 15 |
| 6 | 1 | 00:01:00 | 37 | Buffer | Cont. | 15 |
| 7 | 1 | 00:01:00 | 37 | Buffer | Cont. | 15 |
| 8 | 1 | 00:01:00 | 37 | Buffer | Cont. | 15 |
| 9 | 1 | 00:01:00 | 37 | Acetone 50% | Slope | 20 |
| 10 | 1 | 00:01:00 | 37 | Acetone 75% | Slope | 20 |
| 11 | 1 | 00:01:00 | 37 | Acetone 90% | Slope | 20 |
| 12 | 1 | 00:02:00 | 37 | Acetone | Slope | 20 |
| 13 | 1 | 00:02:00 | 37 | Acetone | Slope | 20 |
| 14 | 1 | 00:03:00 | 37 | Resin 3:1 | Cont. | 10 |
| 15 | 1 | 00:03:00 | 40 | Resin 1:1 | Cont. | 10 |
| 16 | 1 | 00:03:00 | 45 | Resin 1:3 | Cont. | 10 |
| 17 | 1 | 00:03:00 | 50 | Epon | Cont. | 12 |
| 18 | 1 | 00:03:00 | 50 | Epon | Cont. | 12 |
| 19 | 1 | 00:03:00 | 50 | Epon | Cont. | 12 |
| Total time: | 00:49:20 | |||||
