Tools
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Diamond Knife Handling
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.
Diamond Knife Washer
EMS70605 Diamond Knife Washer Instructions Instructions for use: Put the knife wash on a Magnetic Stirrer. (make sure there is NO heat) Fit the Diamond Knife into the holder and tighten the screw making sure that the screw meets the rear of the boat. Fill the wash to the indicated line with distilled or deionized water (you may add two drops of very mild liquid detergent). Put the lid on the container (be careful when doing). Turn the magnetic stirrer on at a low speed for 5 minutes. Remove lid and take knife out. If desired and/or you used detergent, rinse the knife with distilled water. Either let the knife dry naturally or if you prefer you can dry it with a foam rod (polystyrene) dipped in absolute ethanol as usual.
Microtome Blade Guide
When aiming for excellent sections and great blade durability, it is important to match blades to the job. At high performance levels, blades are good for fairly specific tasks for example: 'Select' for 2µm sections of soft tissue, but with little durability Feather R35 is similar to our 'Select' or 'Silver' blades 'Silver' for 2‐4µm sections and improved durability 'Extremus' for 4µm and thicker sections, excellent durability 'Diamond' is for hard materials, even small bones 'Gold' is particularly useful for cryo sections See table at the bottom of the page for Feather® blade comparisons. Disposable Blades For technicians who have never used disposable microtome knives, or for those who occasionally experience problems, the following tips may be helpful. Holder The disposable microtome knife holder is the single largest factor in optimising the results of a disposable knife. If the disposable knife is not adequately secured, vibrations will occur and poor section quality will result. Secondly, a holder that becomes damaged (the slightest nick at the leading edge of the holder, front or back) or has a build up of residue paraffin inside the holder, will limit the holding capability and affect cutting performance. Clearance Angle A change in holder or disposables may require an adjustment to the microtome clearance angle setting. This setting may range between 5 and 12 degrees. Trial and error is the best method for determining your optimum angle. Embedding When embedding, use slightly smaller pieces of tissue. If a larger piece is used, embed the tissue at an angle. Mount hard tissue at the upper portion of the specimen block. Tissue Preparation Pre-chill your block in a mixture of ice and water (slush) before cutting. The combination effects of cooling and moisturising minimises or eliminates compression. Especially before cutting hard tissue, moisturise the face of the tissue for approximately five seconds with a compress of warm water, then cool the block with a tissue-freezing aerosol. These tissue preparation procedures will definitely improve section quality. Technique A slightly slower and more consistent microtome speed is required when using disposables. Poor section quality and irregular tissue dimension are evident if speed is too rapid. Compression Myth It is a myth that a compressed ribbon is evidence of a distorted tissue section. Although severely compressed ribbons may yield unsatisfactory results, it is also true that the finest tissue sections result from ribbons that are deemed overly compressed. The miracle wonders of the warm water bath process soften and flatten compressed ribbons into perfect tissue sections, without detectable distortion in tissue size or cell disruption. Water Bath Temperature A temperature setting of 40 to 42ºC is recommended, especially if the tissue is ultra-thin. Higher bath temperatures may dissolve the paraffin, making it difficult to mount the tissue on a slide. Edge Quality Variations in edge quality are not uncommon for disposables manufactured today. Variables such as microscopic defects in steel, the high speed production processes itself and the frailty of human judgement is sometimes difficult, if not impossible, to control. Accordingly, some edges may require a short break-in period to achieve the perfectly honed edge, while others may simple require a small shift in cutting position of the knife. Profile Descriptions Low Profile Disposable: Used as a general purpose knife: for cutting paraffin sections. It is dimensionally smaller than a high profile knife and is generally sold with a coated edge. It only fits in a low profile holder. Usual dimensions are 76 x 8 x 0.25mm. High Profile Disposable: Used as a general purpose knife: for cutting paraffin sections. It is slightly heavier and higher than a low profile knife and is sold either as a coated or uncoated edge. It only fits in a high profile holder. Usual dimensions are 76mm x 14mm x 0.3mm. Edge Types Uncoated: The edge facets have not been coated. As a result, cutting performance is somewhat limited and cutting resistance and tissue compression may be experienced. However, for may applications uncoated edges perform very well and at an economic price. Uncoated edges perform better if a heavier duty disposable knife is used. Coated: The edge facets have been coated with an edged enhancing substance such as teflon®, polymer, ceramic or titanium nitride (TiN). The edge enhancement reduces the coefficient of friction, making the edge more lubricious. Tissue slides off the cutting edge easily, TiN specifically increased the longevity of the edge. Feather® Blade Comparison Table: Type Description Material / Finish Dimesions (L x W, thick) Applications Soft Tissue Hard Tissue Thin Section Ribbon Section Cryostat LOW PROFILE S35 Standard blade with superior sectioning quality from the beginning. For thin sectioning of paraffin embedded tissue blocks. Stainless steel / Resin coating, platinum sputtering 80 x 8mm, 0.254mm. Edge angle 35° Best - Good - Good R35 Especially suited for Ribbon sectioning. Double spattering increases blade edge strength and durability. For thin sectioning of paraffin embedded tissue blocks. Stainless steel / Resin coating, platinum sputtering 80 x 8mm, 0.254mm. Edge angle 35° Good Best - Best - A35 For extra thin sectioning of paraffin embedded tissue blocks. Especially suited for fibered tissues and biopsy. Stainless steel / Resin coating, platinum sputtering 80 x 8mm, 0.254mm. Edge angle 35° Good Best - Good Good N35 Especially suited for hard tissues. For thin sectioning of paraffin embedded tissue blocks. Stainless steel / Resin coating, platinum sputtering 80 x 8mm, 0.254mm. Edge angle 35° Good Best - Good - N35HR Especially recommended for research laboratories. For thin sectioning of paraffin embedded tissue blocks. Stainless steel / Resin coating, platinum sputtering 80 x 8mm, 0.254mm. Edge angle 35° - Best - Good - S22 For extra thin sectioning of paraffin embedded tissue blocks. Especially recommended for research laboratories. Stainless steel / Resin coating, platinum sputtering 80 x 8mm, 0.254mm. Edge angle 22° Best - Best - - A22 For extra thin sectioning of paraffin embedded tissue blocks. Especially suited for fibered tissues and biopsy. Stainless steel / Resin coating, platinum sputtering 80 x 8mm, 0.254mm. Edge angle 22° Best - Best Good - HIGH PROFILE FHP01 Especially suited for ribbon sectioning. For thin sectioning of paraffin embedded tissue blocks. Stainless steel / Resin coating, platinum sputtering 75.1 x 14mm, 0.31mm. Edge angle 35° Best Good Best Good - FHP02 Durable blade for routine sectioning. Metal surface hardening treatment offers superior blade life. For thin sectioning of paraffin embedded tissue blocks. Stainless steel / Resin coating, platinum sputtering 75.1 x 14mm, 0.31mm. Edge angle 35° Good - Best Good - CRYOSTAT C35 Carbon steel blade for cryostat sectioning. Less curling of sections for easy operation. For thin sectioning of frozen tissue blocks. Carbon / - steel 80 x 8mm, 0.254mm. Edge angle 35° - - - - Best Triple sharpening increases blade durability:
Selecting a Diamond Scribe
The larger the tip/shaft diameter and the greater the included angle the more robust the scribe. Even the heavy duty shaft diamond point tools are meant for scribing light lines for marking or scoring for breaking. They are not meant for engraving or machining. The most robust diamond scribe is with a 90 degree included angle giving the diamond a 1mm base width. It is suitable for students and the general public. It has been purchased for scribing wedding bowls and silver trays by guests. For scribing to break the Deluxe Diamond Scribing Pen is ideal for a production environment where many people may be using the tool. It is more tolerant of untrained personnel who might over apply pressure that would break a more delicate tip. For personal use a more delicate scribe with a finer tip but a sturdy shaft and broad base (90° included angle) would be depending on the scribe head angle desired. Next would be a 60° angle for very delicate scribe to mark or break. Breaking of flat substrates is best facilitated using Wafer Cleaving/Glass Breaking Pliers. The Fine Point Scribes and Micro Tool Scribes are for very delicate scribing like marking identification on the back of a Silicon wafer or glass slide. We don't recommend these for scribe to break applications where a deeper scribe mark may be required. That stated, we can't guarantee that a specific scribe will be suitable for varied applications.
Wand Plastic Properties
Properties of some high technology plastics used in some wands. PEEK Polyetheretherketone General Description PEEK is a high temperature, high cost , semi-crystalline material with excellent mechanical properties and chemical resistance. Brand Names Victrex (ICI), Thermocomp (LNP) Material Mfg. ICI, LNP Tensile Strength Flexural Modulus Impact Strength Maximum Temp. Chemical Resistance Cost 14,000-41,000 psi with carbon fibre 440,000-2,400,000 psi 0.9-3 ft-lb/in notched izod 600F short duration 480F long term Good to Excellent VERY expensive PEI Polyetherimid (Ultem) General Description PEI is an amorphous, high temperature material with relatively low cost compared to other high temperature materials. It has excellent elongation and Impact Strength, and can be molded to tight tolerances. Its chemical resistance is not as good as crystalline materials but is excellent for an amorphous material. PEI behaves similar to polycarbonate, but can perform at higher temperatures. Brand Names Ultem (GE) Material Mfg. GE Tensile Strength Flexural Modulus Impact Strength Maximum Temp Chemical Resistance Cost 14,000-28,000 psi 480,000-1,300,000 psi 2ft-lb/in notched izod 25ft-lb/in un-notched 420F short duration 375F long term Fair to Good Expensive PES Polyethersulfone General Description Polyethersulfone is a high temperature amorphous material with relatively low cost. PES has slightly lower capabilities than PEI at about the same cost. As a result it is not widely used. Brand Names Thermocomp (LNP), Victrex (ICI) Material Mfg. LNP, ICI Tensile Strength Flexural Modulus Impact Strength Maximum Temp. Chemical Resistance Cost 9,500-18,000 psi 590,000-1,200,000 psi 1.4ft-lb/in notched izod 400F short duration 350F long term Good to Excellent Expensive PPS Polyphenylene Sulfide General Description PPS is a high temperature semi-crystalline material. It has good mechanical properties and excellent chemical resistance at elevated temperatures. PPS has been compounded extensively and many different types of properties are available. PTFE filled PPS is one of the best bearing materials available. Unfilled grades of PPS have poor properties, so components are usually made from glass filled or glass/mineral filled grades. PPS is very sensitive to molding conditions and must be processed properly to achieve its maximum potential. Brand Names Fortron (Hoechst Celanese), Lubricomp (LNP), Ryton (Phillips), Supec (GE) Material Mfg. GE, Hoechst Celanese, LNP, Phillips Tensile Strength Flexural Modulus Impact Strength Maximum Temp. Chemical Resistance Cost 14,000-28,000 psi 550,000 -2,900,000 psi 0.5-6ft-lb/in notched izod 500F short duration 450F long term Good to excellent Variable