Ultramicrotomy
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Ultramicrotomy and cryo-ultramicrotomy (Author: Prof. Dr. G. H. Michler)
General information
Ultramicrotomy has developed into an important microscopic examination technique for the preparation of various materials, with highly sophisticated equipment now available. The preparation technique for the successful production of thin and ultra-thin sections, as well as an overview of problems and errors and how to eliminate their causes, is explained by Michler in [1, 2].
Preparation technique
An ultramicrotome is a sensitive cutting instrument in which a sample or test piece is passed over a fixed glass or diamond blade (Fig. 1). The sample arm, with the sample holder and sample attached, is moved towards the blade by a specific, adjustable amount during each cycle.
| Fig. 1: | Schematic representation of the relative movement of the sample and knife during the cutting process (A – feed, B – retraction of the sample arm) |
During the cutting process (between points B and C2 in Fig. 2), a cut is made in the sample with a thickness equal to the feed rate. The exact cutting thickness is determined not only by the set feed rate, but also by other parameters such as the type, quality and angle of the blade, as well as the sample material and the cut size. Cutting is performed at a cutting speed that can be adjusted over a wide range and must be optimised depending on the sample material and the cutting geometry. The sample is returned (D) at a velocity dependent on the cutting speed in such a way that the time for a complete cycle (A1 – C1 – D) remains constant.
| Fig. 2: | Schematic representation of the speed variation of the sample holder arm during the cutting cycle |
Ultramicrotomes can be equipped with either a thermal or mechanical feed mechanism for the sample arm. The thermal feed is based on the physical principle of thermal expansion. The holder arm is fixed on one side so that when it is heated, the expansion only affects the side of the fixed specimen, i.e. in the direction of the blade. The disadvantage of this technique is that the consistency of the cutting process is more or less disrupted with every change in the heat supply due to the inertia of the expansion system. Newer devices of this type have an automatic feed control function that maintains a constant feed rate when the cutting process has to be stopped to remove the sections, etc.
| A1 – A2 | high speed |
| A2 – C2 | set cutting speed |
| B | start of cutting process |
| C2 | end of cutting process |
| C2 – C1 | high speed |
| D | automatic retraction of the sample arm to the starting point |
| A1 | feed (desired cutting thickness) and start of the new cutting cycle |
The mechanical feed used in modern devices is based on a precision mechanical system with a stepper motor. The mechanical feed can be adjusted precisely using digital control, meaning that, in theory, cutting thicknesses from 0 µm to several micrometres are possible. However, the actual cutting thickness depends on the material being cut, and the parameters set.
The resulting sections are usually collected in a liquid-filled trough of the knife, then picked up on carrier nets and examined under a microscope.
Modern devices are equipped with video cameras for demonstration purposes and computers for documentation, e.g. of the cutting parameters.
Application of cryo-ultramicrotomy
Important for fast and clean work is the simple and quick conversion of the ultramicrotome to a cryo-ultramicrotome by placing the cooling chamber on the housing shell. This allows the sample and blade to be cooled separately. While the sample cooling can be set to up to approximately –185 °C depending on the material, less cooling is sufficient for the blade. If flushing fluids (e.g. alcohol) are to be used, their freezing point limits the knife temperature. When cutting with alcohol as the wash-off fluid, it must be taken into account that, due to the low surface tension of alcohol, the cuts sink and can also be strongly compressed. A cryotransfer device is required for the examination of biological samples or moist polymer samples, even in a frozen state, in an electron microscope.
Cutting at lower temperatures is necessary or advantageous if the materials to be cut are too soft and can be hardened or fixed by cooling (e.g. in the case of polymers by cooling to a few tens of K below their glass transition temperature). Cooling takes place in special cooling chambers as attachments for the ultramicrotomes using liquid nitrogen down to –150 °C or –180 °C. In addition to normal glass blades, special cryogenic diamond blades can be used for wet and dry cutting.
After trimming the test specimen, the sample and the knife are placed in the cooling chamber. It is necessary to wait until the sample and the knife have reached the set temperature. The sample and knife can be cooled separately, whereby the knife requires less cooling. When using collection or wash-off liquids, the knife temperature is limited. When using p.a. (pure analysis) alcohol, it can be as low as –55 °C. Even lower temperatures can be achieved with a DMSO (dimethyl sulfoxide) and water mixture.
Cuts of the desired thickness can then be made. If the samples to be cut do not yet have the required hardness, the sample temperature must be lowered further.
With cryosectioning, it is no longer possible to check the section thickness using interference colours. With ultramicrotomes with thermal feed, the only options are empirical values and subsequent checking under a reflectance microscope. The use of devices with mechanical feed is advantageous here.
Acknowledgements
The editors of the lexicon would like to thank Prof. Dr. rer. nat. habil. G. H. Michler, Martin Luther University Halle-Wittenberg, and Polymer Service GmbH Merseburg for their guest contribution.
See also
- Microtomy
- In-situ ultramicrotomy
- Electron microscopy (EM)
- Energy dispersive X-ray scattering (EDX)
- Environmental-SEM (ESEM)
- Velocity
References
| [1] | Michler, G. H., Lebek, W.: Ultramikrotomie in der Materialforschung. Carl Hanser, Munich (2004) pp. 68–71 and 110–112 (ISBN 3-446-22721-0; AMK-Library under F 5) |
| [2] | Michler, G. H., Henning, S., Lebek, W.: Ultramikrotomie in den Material- und Biowissenschaften. Carl Hanser, Munich (2026); 2., revised Edition pp. 69–72 and pp. 115–130 (ISBN 978-3-446-48302-6; see AMK-Library under F 35); https://doi.org/10.3139/9783446484870 |