Brendan Choat, Nele Schmitz
Sectioning is the process of making thin slices of plant tissues for anatomical observation with a microscope. Sections can range from hundreds of microns to tens on nanometers in thickness depending on the target of observations.
- Sliding microtome (sledge microtome): the apparatus used for microtomy, composed of a fixed sample holder and a knife holder that can slide over the sample to make sections. Most commonly used to make sections of hard material such as wood for light microscopy.
- Rotary microtome: the apparatus used for microtomy, composed of a fixed knife holder and a sample holder that can rotate parallel to the knife to make sections. Used to make sections
- Ultra microtome: device to make thin and ultra thin (sub micron) sections. These microtomes employ glass and diamond knives and are commonly used to make sections from transmission electron microscopy.
- Vibratome: type of microtome that uses vibrating razor blade to cut soft tissue. Commonly used to section fresh tissue for light microscopy.
- Cryostat: specialised microtome used to make thin sections while maintaining the sample at freezing temperatures.
- Infiltration: the procedure of infiltrating small or partly degraded samples with a stabilizing medium before embedding and microtomy.
- Embedding: the procedure to stabilize small or partly degraded samples in a medium soft enough to cut with the microtome but dense enough to keep the sample in place. See tissue preparation and embedding summary for details.
Hand sectioning provides a simple technique for making sections of plant material without requiring time consuming fixation and embedding and protocols. Many, if not most fresh plant tissues can be hand sectioned in this way. Particularly good materials are fleshy stems, leaves and roots which are easy to hold while sectioning. Even fairly hard material such as wood can be hand sectioned. In this case, the aim is to cut a small piece of the hard tissue which has been sectioned thinly enough to obtain useful anatomical information. Otherwise, for very soft tissue, use support material around the tissue.
The majority of thin sections are produced with the aid of microtomes. The devices come in a range of shapes and sizes, differing type of tissue that are best suited to section and the thickness of sections they designed to make. Although some hard plant material can be sectioned without embedding, samples cut with a microtome are usually embedded in wax or resin to stabilise the tissue during sectioning.
Microtomy for light microscopy
There are two main types of microtome: the sliding or sledge microtome and the rotary microtome. When it is important to observe the cellular anatomy of the biggest sample possible, the sliding microtome is used for sectioning. A sliding microtome can hold samples of 10 mm or more and is operated with steel knives that produce slides of good quality within a range of 10-30 μm thick. It is therefore mainly used to make thin sections for light microscopical applications.
When using a sliding microtome it is not necessary to embed samples in a stabilising medium. Only when samples are too small to clamp firmly in the sample holder or when samples are too soft or brittle to clamp without distorting them embedding is advised. Not all embedding media can be cut with steel knives. Paraffin and PolyEthyleneGlycol (PEG) are the most commonly used. They dissolve in water and alcohol and therefore don’t require special protocols for section staining and slide preparation.
When a manual sliding microtome is used the distance between the sample and the knife, and thus the section thickness, is regulated by hand. With the newer automatic sliding microtomes, section thickness is controlled automatically by moving the sample holder up with a user-defined distance with each move of the knife holder over the sample.
A rotary microtome is used for sectioning: (1) when thickness of the section is more important than the size of the sample, or (2) when series of sections are of interest. When samples are embedded and cut with a rotary microtome cut sections stick together in a row making it possible to study the anatomical change in axial or radial direction (depending on how the sample was clamped in the holder).
Rotary microtomes have a wider scope of applications since they can be used with steel knives but also with glass or diamond knives. The drawback of using glass or diamond knives is the parallel decrease in sample size. Samples are in general only 2-3 mm3 but can be cut in sections of only a few tenths of a μm thick (glass knife) or even a few tens of a nm (diamond knife) for electron microscopical analyses.
Microtomy for electron microscopy
For observations with the electron microscope sections are generally 60-200 nm thin and are made on a ultra microtome using a glass knife (100-200 nm) or a diamond knife (60-90 nm) for even better quality.
Since sections are so thin and small good preparatory procedures are essential to be able to make quality sections. Therefore it is advisable to take your time for trimming, infiltrating and embedding samples to avoid spending lots of time with the microtomy work and having suboptimal sections. Below a list of causes of having difficulties when cutting:
- Samples are too big
- Poor infiltration of the sample with embedding medium
- Incorrect orientation of the sample in the mould
- Embedding medium of poor quality
For hard samples it is especially important to keep sample size small to allow the thinnest sections to be cut. Mostly the cutting surface of the samples is trimmed to 2 mm2 with the length of the sample somewhat longer (3 mm) to allow correct orientation of the samples in the moulds for embedding after infiltration procedures.
Poor infiltration of the sample with embedding medium can cause holes in the section or sections to be squeezed.
The better the cutting surface of the sample is orientated in reference to the cutting surface of the embedding medium cast, the less you have to change the angles of knife and sample holder to correct for this and thus the easier the cutting.
An embedding medium of poor quality shatters instead of cutting smoothly making microtomy hardly possible.
Since sections are so small and thin fewer steps are required for slide preparation. Glass knife sections can directly be put on a slide with a drop of staining solution and dried on a heating table to affix the section to the slide. Fragile diamond knife sections are not placed on slides but fished out of the water basin with special grids (different grid sizes available) that can directly be placed in the Transmission Electron Microscope.
Health, safety and hazardous waste disposal considerations
When doing microtomy work one should be aware of:
- Handling sharp knives
- Using toxic, corrosive and/or carcinogenic mounting, infiltrating and embedding media
- Using a heating plate with a fire risk if left unattended
- See Microtomy manual for chemically fixed or air-dried wood samples