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What is the Ultramicrotome?
Microtomes are specifically designed devices for the cut of extremely thin sections from samples, for the study of their microscopic composition. Samples can be of biological origins, such as tissues, cells, and tumors, but are not limited to it. Plastics and soft metals can also be processed in the microtome for microscopic evaluation.
However, when the goal is to study the ultrastructure composition of a given sample, the best approach is to analyze it by transmission electron microscopy (TEM), for which an ultramicrotome is the best suitable option for sample sectioning.
Figure 1 – The ultramicrotome A. General composition of a microtome, and respective components (adapted from Atlas of plant and animal histology); B. example of an ultramicrotome currently available on the market, with automatic functions to regulate sample and blade position, and touch screen to regulate additional parameters during sectioning (image credit: Leica Microsystems).
What is Transmission Electron Microscopy (TEM) and Why Does it Need a Special Microtome?
TEM is a specific type of microscopy used to study the ultrastructure of samples, of biological origin or not. The image resolution is defined as the minimal distance between two points in an image that enables their identification as two separate points (Pawley, 2006). Common light microscopy, including confocal microscopy, has a maximum resolution of 0.2 µm (Pawley, 2006). TEM image resolution can be 5000 times superior, achieving 42 picometers (pm) with the most advanced microscopes, meaning that it allows the visualization of much smaller individual sample components than other types of microscopy (Reiner & Kohl, 2008).
In TEM, a beam of electrons passes through (is transmitted to) the sample, producing an image (Figure 2). Therefore, to produce sections so thin that they can be traversed by a beam of electrons, samples must be cut with the ultramicrotome, which can produce sections as thin as 50 nm, the equivalent of a human hair divided into 2000 sections through its diameter. Click here for a more detailed description of TEM and general sample preparation protocol.
In the ultramicrotome, samples are pulled towards a sharp knife (which can be made of glass or diamond), in intervals of up to a few nanometers. The ultracryotome is a special type of ultramicrotome, which is used to cut frozen samples when certain molecular features must be preserved for analysis, and the samples cannot be fixed by chemical reagents.
Figure 2 – Transmission electron micrographs. A. Maturing sheep oocyte (image credit (Karen Reader); B. Several peripheral myelinated fibers and a Schwann cell in the center (image credit: (Fields, 2019).
The fundamental parts of any microtome, including the ultramicrotome, are the sample holder, the knife, and the mechanical mechanism that determines the sectioning interval, i.e., the thickness of the sections, and includes the drive wheel and the knife wheel (see Figure 1).
This is essential in any ultramicrotome since the final sections are impossible to see by the naked eye. The optical system helps the operator to correctly position the specimen and the blade and to adjust the water level for section collection.
During any (ultra)microtomy protocol, the sample must be held into a fixed, rigid material, such as a paraffin block, or embedded in epoxy, an extra-rigid resin in which TEM samples are embedded prior to cutting. The sample holder contains an in-built system to orient the sample and set it in the appropriate cut position relative to the blade (Figure 3).
Figure 3 – Correct and incorrect alignment of the sample block face to the knife edge. The knife should be set at a clearance angle of 5-6° (image credit:(Hughes)).
Blade and water bath:
Blades composed of different materials have distinct applications. For TEM, industrial-grade diamond blades are used to obtain extra-thin sections. The angle position of the blade is also a determinant of the smoothness and thickness of the cut, and thus (ultra)microtomes are also equipped with blade holders that can be adjusted to the appropriate blade position. To avoid friction between the knife and the sample, which could damage the sample, the knives are soaked in water. After sectioning, the slices of material slip to the water bath, where each individual piece can be separated by and collected by a mesh grid (Figure 3).
Wheels (drive wheel and blade wheel):
The two-wheel systems of the ultramicrotome are the main determinants of sample and blade positioning during the cut. The correct adjustment of both wheels determines the smoothness and the thickness of the cut. The smoothest and the most accurate the sectioning is, the lesser the probabilities that artifacts are generated, which may hinder microscopic observations. At the beginning of any ultramicrotomy protocol, the systems are adjusted so as to generate thicker sections of the sample, which are used to set up the region of interest. After, with the aid of the optical system, the blade and sample are positioned to obtain ultra-thin sections for further processing.
Figure 4 – Slice collection after sample sectioning. Upon cut, slices slip onto a water bath, where they can be separated into individual pieces and collected by a mesh grid, where they will be further processed for TEM (Image credit: Atlas of plant and animal histology).
HOW TO HANDLE AND HOW TO MAINTAIN THE ULTRAMICROTOME?
When operating an ultramicrotome, the general rule, as with other laboratory devices, is to follow the manufacturer’s instructions, for handling and care of the instrument. However, some parts of the ultramicrotome are more subject to hand manipulation than others. This is the case of the ultramicrotome knives, which can swap between uses. While diamond knives are highly durable, they are also exceptionally fragile, which is why they require special attention for their long-term duration and function. To improve the duration of diamond knives, it is recommended to; (Guide to Diamond Knife Use & Care, Diamatrix Diamond Knives)
- Store the knife in a closed clean box, whenever it is not being used;
- Not use random areas of the knife blade to cut. Start cutting, by using the edge of the blade. When this area can no longer generate quality sections, proceed to the next segment of the blade. The previous segment can be used to cut thicker sections to rapidly get to the areas of interest of the sample block. This section of the knife can also be used to experiment the cut of new samples, that have not been tested before so that the operator can evaluate how the sample and the blade react during the cut (for example, to evaluate friction between the sample and the blade);
- Only cut sections with the maximum recommended thickness for each blade. Cutting thicker sections requires excessive cutting force, which might damage the edge of the knife;
- Do not allow sections to dry on the knife’s edge;
- Do not change the position and angle of the blade during sectioning, as doing so might critically damage not only the knife but also the sample;
- After using it, the knife must be appropriately clean, according to the manufacturer’s instructions. Distilled water, ethanol or 0.1% Triton X-100 are usually part of the recommendations;
- Re-sharpen the knife when it is no longer able to provide clean cuts. Knife re-sharpening must be performed by specialized operators; this service is usually provided by the seller.
LIMITATIONS OF THE ULTRAMICROTOME
While the ultramicrotome significantly improved our ability to go deep into the nano-world, its use does not come free of limitations. The main limitation of this device is the generation of artifacts, which seems to be directly related to defective maintenance and handling by the operator (Figure 5). The second disadvantage of the ultramicrotome is that to date, this is still an expensive piece of equipment, which requires specialized maintenance, which also presents expensive costs. Thus, only experienced and trained users must work with the ultramicrotome, to ensure long-term duration and function of the device and its parts.
Figure 5 – Transmission electron micrographs showing examples of artifacts resulting from poor ultramicrotome handling. A. Section of rat’s liver showing scratches caused by a damaged knife-edge (image credit: microscopy Australia); B. TEM micrographs of tissue sections showing debris (arrow) and knife marks (dotted arrows; image credit: Monikandan, Joseph, & Rajendrakumar, 2016).
The ability to cut thin sections of material for microscopic observations is a huge contribution to our understanding of the microscopic world. With the development of transmission electron microscopy (TEM), nanometer-thick sections were necessary, as TEM relies on the ability of electron beams to cross the sample to produce an image. To produce such thin sections, researchers use the ultramicrotome, which can cut sections as thin as 40 nm of a wide range of samples. The cut is performed with extremely sharp knives, which are made of glass or diamond. The type of material to be cut determines the type of knife to use, which must be handled with care.
As with other types of microtomes, sectioning artifacts are a limitation of the ultramicrotome, which usually results from poor handling or cleaning of the device. Given that the ultramicrotome is still an expensive piece of equipment, it is not available in all laboratories. Its maintenance can also be expensive, and thus, must only be used by experienced operators.
- Guide to Diamond Knife Use & Care.
- Fields, D. (2019). What is Transmission Electron Microscopy? News Medical Life Sciences.
- Hughes, L. Ultramicrotomy for Electron Microscopy. Retrieved Feb-2020, from https://bitesizebio.com/43238/ultramicrotomy-for-electron-microscopy/
- Karen Reader, K. Transmission electron microscopy techniques. Retrieved Feb-2020, from https://otago.ac.nz/omni/electron-microscopy/tem-techniques.html
- Monikandan, V. V., Joseph, M. A., & Rajendrakumar, P. K. (2016). Studies on artefacts induced in the specimen preparation routines of electron microscopy characterization. IOP Conference Series: Materials Science and Engineering, 149, 012016. doi: 10.1088/1757-899x/149/1/012016
- Pawley, J. B. (2006). Fundamental Limits in Confocal Microscopy. In J. B. Pawley (Ed.), Handbook Of Biological Confocal Microscopy (pp. 20-42). Boston, MA: Springer US.
- Reiner, L., & Kohl, H. (2008). Transmission Electron Microscopy (fifth ed.): Springer.