DiATOME Ultra Sonic Oscillating Diamond Knife

For many years Diatome diamond knives have been used successfully for a wide range of room - and low temperature sectioning applications. Major advances in immunocytochemistry, the sectioning of frozen hydrated specimens, semithin sectioning for optical microscopy, as well as the sectioning of hard industrial samples have been realised using Diatome diamond knives. The development of the Static Line II ioniser enabled dry ultramicrotomy of Lowicryls and a considerable improvement in cryosectioning.

Despite these innovations, until now, one major obstacle remained, preventing us from achieving perfect ultra-thin sections: "compression" which we define as the shortening of the section compared to the sample height. The amount of compression depends on various factors including:

• The wedge angle of the knife.
• The hardness of the sample.
• The interaction diamond surface / section surface.
• The section thickness
• The most critical factor is the wedge angle of the knife. It was shown that reducing the wedge angle results in a reduction of compression, hence better preservation of ultrastructure which allows a higher achievable resolution. However, the wedge angle may not be reduced ad infinitum. A further reduction results in a lower cutting edge quality and a considerably shorter life span.

In cryo-ultramicrotomy, compression, expressed as a percentage, was found almost equal to the wedge angle in degrees:

• 45° knife 40-50%
• 35° knife 30-40% 
• An oscillating knife for low temperature applications is in preparation.

In room temperature ultramicrotomy, we have found the following compression factors (section thickness 50nm):

• 10-20% for Epon, Araldite, EM-Bed, and other epoxy resins.
• 12-24% for Lowicryl K4M.
• 10-17% for Spurr's (hard grade).
• 8-13% for LR White (hard grade).

These limitations have stimulated our efforts to develop the oscillating knife.

It has been shown that compression in frozen-hydrated sections is the limiting factor for successful electron topographic analysis. Cryo sections from vitrified samples without compression were achieved by A. Al-Amoudi, LAU, University of Lausanne.

Benefits of Oscillating Diamond Knives

• Thinner sections
• No compression
• Better structural preservation

The DIATOME ultra sonic, the oscillating diamond knife for room temperature ultramicrotomy. It was developed in collaboration with Dr Daniel Studer, Lab. of Anatomy, University of Berne.

A piezo actuator produces an oscillation of the knife at a desired frequency and amplitude, parallel with the cutting edge. A depression in the foot of the knife allows the oscillation parallel to the cutting edge. The depression is rigid in the north/south direction and guarantees stability in the cutting direction.

The new knife produces ultrathin sections almost free of compression.The sections become thinner at the same thickness setting: since the volume of the section remains the same, the increased length leads to a decrease in thickness.

The theoretical considerations and first results of this invention were presented in the Journal of Microscopy. Results with polymers using the oscillating knife were shown at the M&M 2002 meeting in Quebec.

We have tested the oscillating knife with the following samples:

• Biological samples in Epon, Araldite, EM Bed, etc.
• Biological samples in acrylic resins (Lowicryls, LR White).
• Rigid polymers such as PS, PMMA, ABS, HIPS, modified PP, etc.

Peripheral nerve (rat)

Heart (rat)

Human keratinocyte

ABS

Impact modified PP

Specifications

• Knife angle: 35°
• Knife cutting range: 10-80nm
• Knife cutting edge length: 3.0mm

Control Unit

• Frequency: 25-45kHz, or automatic setting of the resonance
• Amplitude: variable (Voltage 0-30V)
• Mains voltage: 230V, 110V

HANDLING

Sample preparation

Trim the sample with a trim 45 or a trim 20 diamond blade. The sample width should be a maximum of 0.5 mm.

Measuring the sample height

With the use of an eyepiece graticule in one of the stereomicroscope oculars (graticule 10450336 for the Leica M80). Sample block (fixed in the sample holder), is mounted in the trimming plate. Measure the height with the graticule.

Ultramicrotome Settings

• Set the clearance angle to 6° as shown on the guarantee card.
• Set the desired section thickness.
• Set the sectioning speed (0.4 – 0.6 mm/sec)
• Tighten all set screws.

Installing the knife

• Mount the knife in the knifestage of the ultramicrotome and tighten the set screw.
• Connect the control unit to the power supply.
• Connect the control unit OUTPUT and the knife with the blue cable.
• Switch on the control unit (switch ON the back side).
• Set the resonance frequency (Toggle switch on AUTO). After a few seconds the display will show «Peak locked».
• Adjust the amplitude to approx. 2 V.
• Approach the sample with the knife (settings as shown in our diamond knife handling manual.
• Start sectioning as usual.

Measuring the section length

• Measure the section length with the graticule and compare it with the sample height.
• If sections are too short, increase the the amplitude (turn the button clockwise).
• If sections are too long, decrease the amplitude (turn the button counterclock wise).
• Too high an amplitude may lead to drifting of the sections.

Drifting of the sections

When working in resonance, the sections may drift slightly to the right or to the left. If this is the case, the following procedure helps: Switching from AUTO to MAN. Now increase or decrease the the frequency a few hundred Hertz, until the sections float straight on the water surface.

If sections drift to the right: decrease the frequency. If sections drift to the left: increase the frequency.

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Back:

References

1. H. Sitte: Advanced Instrumentation and Methodology related to Cryoultramicrotomy: a Review. Scanning Microscopy Supplement 10, pp. 87-466, 1996.
2. M. Michel, H. Gnägi and M. Müller: Diamonds are a cryosectioner's best friend. Journal of Microscopy, Vol. 166, Pt 1, pp. 43-56, 1992.
3. O.L. Reymond: The diamond knife "semi": a substitute for glass or conventional diamond knives in the ultramicrotomy of thin and semi-thin sections. Bas. Applied Histochemistry, No. 30, pp. 487-494, 1986. Various publications on materials ultramicrotomy. Microscopy Research and Technique, Vol. 31. Number 4, pp. 265-310, 1995.
4. L. Edelmann: Freeze-substitution and the preservation of diffusable ions. Journal of Microscopy, Vol. 161, pp. 217-228, 1991.
5. J.C. Jésior: How to avoid compression. Journal of Ultrastructure and Molecular Structure Research, pp. 210-217, 1986.
6. J.C. Jésior: Use of low-angle diamond knives leads to improved ultrastructural preservation of ultrathin sections. Scanning Microscopy Supplement 3, pp. 147-153, 1989.
7. K. Richter: Cutting artefacts on ultrathin cryosections of biological bulk specimens. Micron, Vol. 25, No. 4, pp. 297-308, 1994.
8. K. Richter, H.Gnaegi and J. Dubochet: A model for cryosectioning based on the morphology of vitrified ultrathin sections. Journal of Microscopy, Vol. 163, Pt 1, pp. 19-28, 1991.
9. C.E. Hsieh, M. Marko, J. Frank and C.A. Mannella: Electron tomographic analysis of frozen-hydrated tissue sections. Journal of Structural Biology 138, pp. 63-73, 2002.
10. J.R. McIntosh: Electron Microscopy of Cells: A new beginning of a new century. The journal of Cell Biology, Vol. 153, pp. 25-32, 2001.
11. A. Al-Amoudi, J.Dubochet, H. Gnaegi, W. Lüthi, D.Studer: An oscillating cryo-knife reduces cuttinginduced deformation of vitreous ultrathin sections. Journal of Microscopy, Vol. 212, Pt 1, pp. 26-33, 2003.
12. D. Studer and H. Gnägi: Minimal compression of ultrathin sections with use of an oscillating diamond knife. Journal of Microscopy, Vol. 197, Pt 1, pp. 94-100, 2000.
13. J.S. Vastenhout and H.Gnaegi: Ultramicrotomy of polymers using an oscillating knife; improving polymer morphology. Microscopy and Microanalysis. 8 (Suppl. 2) 2002.