TOOL - Diamond Knife Handling

Created by Karen Darley, Modified on Mon, 13 May at 1:32 PM by Kathleen Patrick

Diamond Knife Handling

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 diamond knife can withstand a great deal of use. However, 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.
Ensure that the receiving department of your institute or facility does not open the plastic box for they can cause damage.
Do not disturb the knife edge when unpacking the knife.

Once the knife is unpacked it is ready for use; just place it in the stage and begin sectioning.

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 will 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 Trimtool 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, 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. 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 glass particles sticking on the block.

Sectioning

1. Room Temperature

The following are factors that will determine, in part, the quality of your sections.

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.
Trough liquid: the water should be level with the cutting edge and give a good reflection. 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:

Do not add solvents to the distilled water in the boat (ie, acetone) as they:

may dissolve the sealing material between the knife and the boat.
will reduce the surface tension of the water.
may also dissolve and destroy the cellular materials of your specimen.

Avoid cutting thicker than the limits given for each specific knife.
Ensure the block is fully cured and not 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.

Diamond knives produce higher quality cryo sections than glass knives. With a diamond knife it is possible to cut thinner sections with 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 the specimen that needs to be cut.

Cleaning Procedure

Method 1 - polystyrene cleaning sticks
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 high density polystyrene diamond knife cleaning sticks and bevel it to an angle approximately 45o using an oil free razor blade.
Dip the stick into 100% ethyl alcohol and shake off the excess.
Pass the stick over the cutting edge without applying pressure. Repeat, as necessary, until the edge is clean.

This procedure has been used for many years and we have recommended it to all of our customers. The results have been exceptional with no evidence 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.

This mechanical cleaning is required after cutting poorly polymerised blocks.

Method 2 - non-contact

Immediately after picking up the sections:

Remove all unused sections with a hair curl or an eyelash.
Rinse the knife thoroughly with distilled water.
Blow the water off of the knife using a can of clean pressurised air.

If the knife is tended to immediately after sectioning, there is no need for any more elaborate cleaning.

Method 3: - dried sections and other debris

If sections or debris dry on the knife edge we recommend you:

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.
Proceed to clean the knife using method 1.

For cleaning cryo knives:

Remove the knife from the cryo chamber (before heating the chamber up).
Rinse the knife under tap water to warm it up.
proceed to clean using method 1.

For cryo knives we recommend a 50% solution of ethanol in place of 100%.

TROUBLESHOOTING

1. Problem: Difficulty Wetting the Knife Edge

Solution:

Clean the knife edge with alcohol (100%) and a diamond knife cleaning stick.
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.
pass a diamond knife cleaning stick over the cutting edge (the boat being full and mounted in position) using the same motion as the cleaning method.
pass a hair or eyelash 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 re-cement it for you.

2. Problem: Block Face is Getting Wet

Causes:

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.).

Solution:

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.

For methacrylates:

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. Adjust the light source to the appropriate angle. If your ultramicrotome does not allow for adjustment of the light source:

Tape a small piece of aluminum foil to the light source.
Slowly bend the foil until you reach your desired reflection.

3. Problem: Chatter

Causes:

Chatter can be caused by:

External vibrations.
A faulty microtome.
Screws are not fully tightened (block, block holder, or knife).
Cutting pressure is too great.
Clearance angle is too small (may cause friction between the block face and the diamond face).

Solution:

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

Causes:

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.

Solution:

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

Most diamond knives need re-sharpening due to knife marks. They are found in the cutting edge and cause very fine lines in sections.

Causes:

Touching the edge with fingers or any solid object.
Remnant particles from block trimming.
Hard particles in blocks and specimens.
Normal use of the diamond knife that, over time, will all cause nicks.

Solution:

Keep all hard objects as well as fingers away from the cutting edge.
Follow our recommendations under the trimming section for best results.
Control block inclusions. 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.
have a dedicated knife for each specimen type. Normal wear and tear is inevitable. You can extend the periods between resharpening by using a different knife for different types of specimens.

6. Problems: Striations

Whereas nicks are found on the knife edge, striations are found in the sections itself.

Causes:

Very fine imperfections in the cutting edge.
Poorly polymerised blocks.
non-homogenous blocks.

Solution:

ensure the blocks are fully polymerised.
Change the block.
Try another portion of the cutting edge.