Until recently (Giberson et al., 1997, J. Vet. Diag. Invest. 9:61-67) there were no unified protocols for microwave assisted rapid processing of tissue for electron microscopy. Recent work (unpublished) has demonstrated that the use of vacuum during processing saves time and improves fixation quality over previously established protocols (Giberson et al., 1997).
The table on the previous page shows the effects of Microwaves/Vacuum on the Sample Processing Times for Electron Microscopy
Microwave technology has reduced the times required for sample processing by over 90%, when compared to routine processing protocols. Water recirculation, the temperature probe and variable wattage have made protocol development possible. The recent addition of a microwave vacuum chamber has improved sample preservation and reduced times required for resin infiltration by >80%, over previously established microwave protocols.
Protocol:1. Microwave Oven Calibration:
Figure 1 indicates the placement of the water loads in the microwave cavity and the vacuum chamber during VMP. The combination of water loads shown in Figure 1 creates an excellent cold spot during the fixation and buffer rinse steps. The heating rate for the microcentrifuge tubes should be 8-10°C (when 600µl of water is used - 40 seconds at 100% power under vacuum - 20"Hg).
It is best to determine the actual heating rate with the buffer and fixative combination being used. Osmium will tend to heat less than the aldehydes over the same time interval.
Fig. 1. The finder mat grid is taped to the oven cavity, approximately 1" from the front of the microwave cavity and centered side to side. An 800ml or 1 L plastic beaker (glass is all right but it creates more heat in the oven cavity) is filled with 500-700ml of tap water and the water is recirculated and cooled to under 35°C using the Load Cooler. The vacuum chamber is placed, as shown, in the microwave cavity. It has 2 each 100ml water loads inside which are always oriented as shown. The holder for the sample tubes (A) is placed in front to the two water loads, so that one of the sample tubes is directly below the temperature probe, which is inserted through the O-ring in the top of the chamber (see Fig. 3).
2. Aldehyde Fixation:
Microcentrifuge tubes which contain the specimens and 600µl of fixative are placed in the microcentrifuge tube holder (Fig. 2) which is positioned in the vacuum chamber as shown in Fig.1. A vacuum (20" Hg; 500 torr) is drawn. The microwave is then programmed for the following time sequences (these three time/power intervals are programmed sequentially on one numbered key pad):
- 1 minute at 0% power
- 40 seconds at 100% power
- 3 minutes at 0% power
The starting temperature of the fixative should be ≤20°C.
Fig. 2 The microcentrifuge tube holder is made of PTFE. The volume of fixative in the microcentrifuge tube should be 600µl ±100µl. Variations greater than 100µl will change the heating rate of the fixative solution.
Figure 3 is a side view of the vacuum chamber. The temperature probe is to be positioned about 3mm above the surface of the fixative in one of the sample tubes. At the end of the 40 seconds at 100% power the microwave door is opened and the temperature probe is pushed down into the fixative to record the temperature after microwave irradiation. The temperature change should be >8°C. After noting the temperature, close the microwave door and push the start button. The final 3 minutes of 0% power will count down.
Fig. 3. The temperature probe (C) is positioned ~3mm above the fixative level in one of the microcentrifuge tubes. Beaker "A" is a standard disposable 100ml polypropylene beaker. The 3-way vacuum valve (D) is positioned toward the front of the microwave, the vacuum hose attached and a vacuum drawn (20" Hg; 500 torr). Close the valve. When the temperature probe is pushed down into the fixative to record the final temperature, after microwave irradiation, the vacuum will remain intact.
3. Buffer Rinse:
At the end of the 3 minute 0% power sequence, break the vacuum and remove the holder and microcentrifuge tubes. Remove the fixative and replace with buffer. Immediately remove the buffer and add 600µl of fresh buffer. Place the holder with tubes back in the vacuum chamber, draw a vacuum (20" Hg; 500 torr) and microwave for the following intervals:
- 1 minute at 0% power
- 40 seconds at 100% power
4. Osmium Fixation:
Remove the buffer and add 600µl of osmium fixative. It is best if the fixative temperature is <20°C before starting. Repeat the steps for aldehyde fixation (1 min. 0% power / 40 Sec. 100% power / 3 min. 0% power). At the end of the sequence, remove the osmium from the samples (under the fume hood) and rinse the tissue with water. Make sure to note the temperature after the 100% power cycle. In our experience the osmium step will heat less than the aldehyde under vacuum.
Replace the two 100ml water loads with fresh tap water prior to starting osmium fixation.
5. Water Rinse:
Quickly rinse the samples with tap water prior to transferring them to the flow-through baskets for dehydration (see Giberson, et al., 1997) and resin infiltration.
We typically use acetone in the following concentrations: 1 x 50%; 1 x 70%; 1 x 90%; 2 x 100%. Ethanol can be used in place of acetone if desired. A temperature restriction (37°C) is used during the dehydration steps which entail 40 seconds at 100% power for each step. The flow-through baskets are placed in polypropylene petri dishes and approximately 15-18ml of solution is used for each step (Fig. 4).
The Petri dish with baskets, shown in figure 4, is placed in front of the water load (about 4") serviced by the load cooler and a second water load of about 400ml is place to the right of the petri dish. The temperature probe is placed in the probe stand.
7. Vacuum Resin Infiltration:
The second water load, added for dehydration, is removed as are the two 100ml water loads from the vacuum chamber. The petri dish with baskets is placed in the vacuum chamber. The lid to the vacuum chamber, with the temperature probe inserted, is placed on the top of the chamber (Fig. 3). Make sure the tip of the temperature probe is in the resin in the petri dish. Position the vacuum chamber in the same location that was used for fixation. Set the temperature restriction to 43°C and draw a vacuum (20" Hg; 500 torr). Three two minute vacuum infiltration steps are done in the microwave (100% resin is used for each infiltration step). Use fresh resin for each step. After the last two minute run the tissue is ready for embedding in capsules and polymerization.
The embedding capsules are polymerized under water in the microwave (see Giberson, et al., 1997) (Fig. 5). Since the publication of Giberson, et al., 1997, we have found that the use of the temperature probe during polymerization improves the shape and overall block quality after polymerization.
In figure 5 a 4-cup, rectangular polypropylene dish is used during polymerization (36133) . It will hold approximately 1000ml of water. The large water load (serviced by the load cooler is left in the microwave. Water should be added as needed to maintain a level above that of the embedding capsules.
The following polymerization schedules are recommended for the resins listed below:
a. Epoxies: 10 min. at 60°C; 10 min. at 70°C; 10 min. at 80°C; 45 min. at 100°C
b. LR White: 10 min. at 60°C; 10 min. at 70°C; 25 min. at 80°C
c. Histocryl: 10 min. at 60°C; 10 min. at 70°C; 10 min. at 80°C; ~30 min. at 90°C
The Effect of Microwaves/Vacuum on the Sample Processing Times for Electron Microscopy
mins = minutes
hrs = hours
|Routine M/wave Process.1||Vacuum M/wave |
|1. Primary Fixation (aldehyde)||10 mins||6 mins||1 hour|
|2. Buffer Rinse||6 mins||4 mins||0.5 hrs|
|3. Secondary Fixation (osmium)||10 mins||6 mins||1 hr|
|4. Dehydration (acetone/ethanol)||7 mins||7 mins||2 hrs|
|5. Resin Infiltration||50 mins||8 mins||18 hrs|
|6. Tissue to Embedding Capsules||15 mins||15 mins||0.25 hrs|
|7. Resin Polymerization||45-75 mins||45-75 mins||18 hrs|
|TOTALS||~170 mins||~120 mins||~2,400 mins|
1Giberson, et al., 1997. J. Vet. Diagn. Invest. 9:61-67. 2Unpublished work in progress.