Preparing samples for the electron microscope

Electron microscopes are very powerful tools for visualising biological samples. They enable scientists to view cells, tissues and small organisms in very great detail. However, these biological samples can’t be viewed on electron microscopes whilst alive. Instead, the samples must undergo complex preparation steps to help them withstand the environment inside the microscope. The preparation process kills the tissue and can also cause changes in the sample’s appearance.

For scientists who wish to view biological samples, this poses a challenge – how can the sample be preserved so that it looks as much as possible like it would in the living organism, while still being able to withstand being visualised in the electron microscope.

Surviving a hostile environment

There are two reasons why living things can’t survive in an electron microscope:

• The power of the electron beam that’s directed at the sample.
• The vacuum inside the microscope.

The electron beam inside a transmission electron microscope (TEM) causes problems for biological samples because of its high energy. It needs to have enough energy to pass right through the sample and out the other side. The temperature can get up to 150°C where the beam hits the sample. This temperature is far too high for living cells to survive. Scanning electron microscopes (SEMs) use a lower-energy electron beam, but it can still be damaging to the sample.

The vacuum inside an electron microscope is important for its function. Without a vacuum, electrons being aimed at the sample would be deflected (knocked off course) when they hit air particles. But liquid water, which is abundant in biological samples, evaporates immediately in a vacuum. If this happened, a biological sample would vaporise in front of your eyes!

To be visualised by an electron microscope, biological samples need to be:

• fixed (stabilised) so the electron beam doesn’t destroy them
• dried thoroughly so the vacuum doesn’t affect them.

Right from the word go, from the moment you collect your sample, you have to be thinking about preserving it in as close to the living state as possible.

Allan Mitchell, Microscopy Otago

Fixation: a snapshot of the living sample

The first – and perhaps most important – step in the preparation process is fixation. In this step, living tissue is chemically treated to stabilise it. This kills the tissue sample at the same time. It’s important to fix a sample as quickly as possible because, as soon as tissue is removed from its natural environment, it starts to change. For instance, oxygen levels start to drop as soon as tissue is removed from an organism. This causes mitochondria to start to change their appearance. Another common change in the fixation process is that lipids tend to form micelles.

Looking out for artefacts of fixation

Micelles and strange-shaped mitochondria are examples of artefacts – structures that are seen under the microscope but aren’t found in living cells. It’s very important to be aware that artefacts can be introduced during fixation so that you don’t mistake them for real parts of your sample. Telling the difference between an artefact and a ‘real’ structure can be difficult.

To minimise the introduction of artefacts, scientists are continually experimenting with new ways to prepare samples. One approach is to freeze the sample very quickly instead of fixing it. Providing the sample stays cold enough, this ‘locks up’ the water and prevents it from evaporating inside the microscope. Freezing samples is common in SEM (and is known as cryoSEM). It is still in the early stages of development for TEM.

Sample preparation in TEM and SEM: the differences

Fixation and dehydration are important for preparing samples for both the TEM and the SEM. However, other aspects of sample preparation differ greatly because the two microscopes have different requirements.

For TEM, samples must be cut into very thin cross-sections. This is to allow electrons to pass right through the sample. After being fixed and dehydrated, samples are embedded in hard resin to make them easier to cut. Then, an instrument called an ultramicrotome cuts the samples into ultra-thin slices (100 nm or thinner). TEM samples are also treated with heavy metals to increase the level of contrast in the final image. The parts of the sample that interact strongly with the metals show up as darker areas.

Samples destined for the SEM aren’t cut into thin sections, because the SEM visualises the surface of three-dimensional objects. Instead, SEM samples are coated with a thin layer of metal (usually gold or gold-palladium). The metal coating makes samples conductive. It acts in a similar way to an electrical wire, drawing away the electrons that are bombarding the sample. Without the metal coating, many samples build up electrons, and this can cause ‘charging artefacts’. These are strange-looking areas on SEM images that give a false impression of how the sample looks.