A. Diamond Knife Handling
Contrary to popular belief a diamond knife is not as fragile as you might think. Taking into account the vast array of specimens that it is able to cut successfully from soft to hard, the diamond edge is quite durable. With proper care and handling, the DiATOME diamond knife can withstand a great deal of use. However, with all of this said, in order to avoid any unfortunate mishaps, the following precautions should be taken when receiving the knife:
Make sure that the seal on the outside of the knife box has not been tampered with.
Insure that the receiving department of your institute or facility does not open the plastic box for they can cause damage.
When unpacking the knife, care should be taken not to disturb the knife edge.
Once the knife is unpacked it is ready for use*; just place it in the stage and begin sectioning.
* During our final inspection before packaging, the cutting edge is thoroughly cleaned. Only knives that pass our high inspection standards are released for shipping. Therefore, no cleaning is required by you for your initial use.
WE DO NOT RECOMMEND THE TESTING OF KNIVES WITH BLANK BLOCKS BECAUSE THEY CAN NOT SHOW THE KNIVES TRUE CUTTING ABILITY.
B. Preparation Prior to Sectioning
A perfect section ribbon can only be obtained from a well trimmed block. The upper and the lower side of the block must be parallel to the knife edge. The block should not be too wide because this would substantially increase the cutting pressure. This may result in chatter (see troubleshooting below).
We have found that the best trimming results with plastic embedded specimens are obtained with our Diamond trimming tools. For the trimming of frozen specimens, biological as well as industrial, our cryo diamond trimming blade offers the best results. If trimming is done with a razor blade, always use a fresh degreased blade. Before the final few cuts of your block are made, we highly recommend that you change to a new blade.
Caution: If the razor blade is old or the cutting of the block is too thick, the blade does have a tendency to leave steel particles (shavings) on the block. Upon sectioning, these particles will cause damage to the diamond's edge.
If the block face is precut with glass knives, use only a clean new portion of the edge. This will avoid glassy particles sticking on the block.
1. Room Temperature
The following are factors that will determine, in part, the quality of your sections. These points should be checked prior to you beginning your sectioning.
Alignment of the block: it must be parallel to the cutting edge.
Adjustment of the hardware: All of the screws should be tightened in the block holder, the knife holder, etc.
Knife parameters: The clearance angle and the cutting speed should be set as indicated on the cutting card. (see all exceptions in Troubleshooting).
Trough liquid: the water should be level with the cutting edge and give a good reflection. (see all exceptions in Troubleshooting.) The boat should be filled until the cutting edge is moistened. Then the left and right hand sides of the boat are wetted with the tip of a toothpick without touching the knife edge or the cement mounting material.
To avoid difficulties during sectioning and extend the life of your diamond knife, the following tips should be kept in mind:
Do not add solvents to the distilled water in the boat (ie, acetone) for the following reasons:
- They may dissolve the sealing material between the knife and the boat.
- Solvents will reduce the surface tension of the water.
- Solvents may also dissolve and destroy the cellular materials of your specimen.
Avoid cutting thicker than the limits given for each specific knife.
The block should be fully cured and should not be too wide.
Exercise care when picking up sections. Do not touch the cutting edge with any solid object (grids, loops, tweezers).
Do not allow the sections to dry on the cutting edge.
2. Cryo Temperatures
Cryo sections have a tendency to stick to the knife edge, curl, bunch up one on top of the other, and fly away. These problems are largely caused by electrostatic charging in the cryo chamber. The lower the temperature is, the more pronounced these problems become.
To eliminate these problems and make cryo sectioning as easy as sectioning at ambient temperatures, we highly recommend that you use our "Static-Line" II Ionizer in conjunction with our cryo diamond trimming tool and knife. It has been proven that diamond knives produce higher quality cryo sections than that of glass knives. With a diamond knife it is possible to cut thinner sections and these sections have less compression and greater uniformity than those cut on glass knives. The most widely used technique in cryo sectioning is the Tokuyasu technique. For this technique we have found that the optimum results are achieved when using a diamond knife at the following temperatures:
For trimming and semi-thin sections: - 80°C
For ultra-thin sectioning: -110°C to -120°C
For all other techniques the temperature range will be dependent on your specimen that needs to be cut.
D. Cleaning Procedure
Immediately after picking up the sections, remove all unused sections with a hair curl or an eyelash. Then proceed to clean the knife edge as follows:
Empty the boat and remount it in the ultra microtome support stage.
Take one of our polystyrol rods and bevel it to an angle approximately 45o using an oil free razor blade.
Dip the rod into 100% ethyl alcohol and shake off the excess.
Pass the rod over the cutting edge without applying pressure* You may repeat this procedure, if necessary, until the edge is clean.
This procedure has been used for many years by us and we have recommended it to all of our customers; the results have been exceptional with no evidence at all of knife damage.
It is important not to contact the shaded (cemented) areas as this will damage the edge of the polystyrol rod and transfer dust to the cutting edge of the knife.
MECHANICAL CLEANING AS EXPLAINED ABOVE IS ABSOLUTELY NECESSARY AFTER CUTTING POORLY POLYMERIZED BLOCKS.
If you do not feel comfortable using the method described in procedure 1, there is an alternative. Immediately after picking up the sections proceed as follows:
Remove all unused sections with a hair curl or an eyelash.
Rinse the knife thoroughly with distilled water.
Take a can of clean pressurized air and blow the water off of the knife.
If the knife is tended to immediately after sectioning, there is no need for any elaborate cleaning.
If sections or debris dry on the knife edge we recommend the following procedure be followed:
Place the knife in distilled water.
Add one or two drops of mild liquid dishwashing detergent to the distilled water.
Let it sit overnight.
Remove the knife and rinse it with distilled water only.
Now proceed to clean the knife using method 1.
For the cleaning of our cryo knives proceed as follows:
Remove the knife from the cryo chamber (before heating the chamber up).
Rinse the knife under tap water to warm it up.
Then proceed to clean using method 1.
(Please note: for our cryo knives we recommend a 50% solution of ethanol in place of 100%.
The following is a summary of problems which can occur when sectioning with a diamond knife and how to solve them.
1. Problem: Difficulty Wetting the Knife Edge
Clean the knife edge with alcohol (100%) and a DiATOME cleaning rod.
Fill the boat with distilled water until the water level is a little too high, wait a few minutes, and then carefully remove the excess water.
A DiATOME cleaning rod is passed over the cutting edge (the boat being full and mounted in position) using the same motion as the cleaning method.
A hair or eyelash is drawn over the tongue and then passed over the cutting edge of the knife while the boat is full and clamped in place.
If the cementing material is damaged and you suspect that this is the reason you can not wet the knife edge, please contact us immediately so we may arrange to get your knife back and recement it for you.
2. Problem: Block Face is Getting Wet
For epoxy resins: Block faces may get wet due to electrostatic charging (low room humidity and or transportation).
For methacrylates: Some of these embedding materials are hydrophilic and tend to wet the block surface because they attract water (Lowicryl, LR White, etc.).
For epoxy resins:
Increase the room humidity.
Lower the water level ever so slightly.
Dry the block face with filter paper.
Eliminate electrostatic charging with an antistatic device.
Lower the water level to a concave shape.
Two problems will become pronounced by lowering the water level.
You will have difficulties wetting the edge.
To combat this just follow the steps outlined above in edge wetting.
You will have difficulties with reflection.
To alleviate your reflection problem you should adjust the light source to the appropriate angle.
If your ultramicrotome does not allow for adjustment of the light source proceed as follows:
Tape a small piece of aluminum foil to the light source.
Slowly bend the foil until you reach your desired reflection.
3. Problem: Chatter
Chatter manifests itself in many ways and is caused by different reasons.
A faulty microtome.
Screws are not fully tightened(block, block holder, and knife).
Cutting pressure is too big.
Clearance angle is too small (may cause friction between the block face and the diamond face).
Change the location of the microtome.
Have the microtome checked by a service engineer.
Make sure all of the screws are tightened.
Reduce the block width.
Increase the clearance angle by 1-2 degrees.
4. Problem: Compression
The block is too soft.
The knife angle is too big.
The knife is dull.
The clearance angle is too big and or the cutting speed is too high.
Make the blocks harder.
Switch from the 45 to the 35 degree angle knife.
Send the knife back to us for evaluation and possible resharpening.
Reduce the clearance angle by 1-2 degrees and reduce the cutting speed from 1mm/sec to 0.5mm/sec.
5. Problem: Knife Marks
The majority of all diamond knives that are in need of resharpening are due to knife marks. They are found in the cutting edge and cause very fine lines in your sections.
Causes: Knife Marks:
Touching the edge with your fingers or any solid object.
Remnant particles in your block from trimming.
Hard particles in your block and specimen.
Normal use of the diamond knife that over time anddepending on the different types of specimens will all cause nicks.
Solution: Knife Marks:
Some of the causes are easily prevented while others are more difficult to prevent.
Keep all hard objects as well as fingers away from the cutting edge.
Follow our recommendations under the trimming section for best results.
Most blocks have hard particles in them. These inclusions can be controlled, to an extent, by keeping to the highest possible purity during the entire block making procedure from fixation through sectioning. It is impossible to avoid some inclusions but keep in mind that the knife marks produced are so small that they are not even detectable in a light microscope but only through the sectioning test.
Normal wear and tear is inevitable. You can extend the periods between resharpening by using a different knife for each of your different types of specimens.
6. Problems: Striations
Whereas nicks are found on the knife edge, striations are found in the sections itself.
Very fine imperfections in the cutting edge.
Poorly polymerized blocks.
Make sure the blocks are fully polymerized.
Change the block.
Try another portion of the cutting edge.