How the Scanning Electron Microscope works

Preparing samples for scanning electron microscope imaging

Blog post by Clodagh Dooley

Scanning electron microscopes (SEM) are amazing tools that allow imaging far beyond the resolution capabilities of light microscopes. SEMs work by creating a focused beam of electrons that scans over a sample; interacting with and exciting its atoms and generating signals that can be used to derive an image of the samples topography.

SEM blog

Fig. 1.Sage leaf surface hairs. Images by Dr. Clodagh Dooley, AML, CRANN, TCD.

SEM blog2

Fig. 2. The scanning electron microscope used to image the Trinity Tree samples. The tool is based in The Advanced Microscopy Lab, CRANN, TCD. The microscope is a Zeiss Ultra FESEM with Gemini column and Quorum Cryo preparation chamber.

To avoid scattering of the electron beam, the electron source and the sample chamber are under vacuum. This causes no issue when working with a dry sample, such as a metal or ceramic, but can cause big problems when working with a sample with a high water content. Samples with a high water content, such as plant material, use the water as support for its structure, if this water is removed rapidly, as will happen when placed in a vacuum, then the sample will dehydrate and collapse.

SEM blog3

Fig. 3. The vacuum chamber of the SEM showing the loading stage.

SEM blog4

Fig. 4. The vacuum chamber of the SEM showing the loading stage.

The first step in preparing a biological sample for SEM is to preserve it using glutaraldehyde, a chemical fixative that prevents any breakdown and degradation of the structure. The next step is to deal with the water. This is usually done in one of two ways; removing the water in a slow, controlled manner with help of solvents or solidifying the water as ice and imaging a frozen sample.

In the first method the water in the sample is slowly replaced with alcohol by immersing it in deionised water/alcohol solutions with increasing alcohol concentration until it is in a 100% alcohol solution. The alcohol immersed sample is placed in the critical point dryer (CPD) chamber and the alcohol solution is very slowly replaced with liquid CO2. Once the alcohol within the chamber has been completely replaced with liquid CO2 the system is brought to what is termed as the ‘critical point’ for CO2. This is where the pressure and temperature within the CPD chamber causes the liquid CO2 in the sample to turn from a liquid to a gas. The gas can then be vented off without any distortion of the sample due to surface tension.

SEM blog5

Fig. 5. The Quorum Critical Point Dryer

The second method involves the use of liquid Nitrogen. The sample is plunged into liquid Nitrogen slush (-190°C) to flash freeze. It is then transferred, under vacuum, to a cold stage within the SEM imaging chamber where it is held at this temperatures throughout imaging. With this method of preparation, chemical fixation can be avoided as the flash freezing preserves the sample structure and prevents degradation.

SEM blog6

Fig. 6. Cryo preparation chamber on the side of SEM chamber.

The final step before we can image a biological/hydrated sample is to coat it with an electron conductive coating; commonly used coating materials include carbon, gold, palladium and platinum. Conductive coating prevents charging of the specimen, which is caused by accumulation of static electric fields and makes imaging difficult.

SEM blog7

Fig. 7. Cressington 208 Turbo sample coater.

SEM blog8

Fig. 8. Plant material coated in a 10nm layer of Gold Palladium.

About this blog

This blog celebrates the stunning selection of Trees in Trinity College Dublin.

It also outlines Trinity Trees, an ambitious ongoing project involving the Trinity College Tree specialist (David Hackett), a scientist (Prof. David Taylor), a microscope expert (Dr. Clodagh Dooley) and an artist (Olivia Hassett) all based in Trinity College Dublin.

The team aim to make visible fascinating microscopic elements of the trees. This will allow for an unique way of engaging with the trees in an urban setting.

In response to the research and microscopic imagery the artist will create of a series of innovative art works, which will be installed in specific trees throughout the campus. This exhibition will launch in September, 2017. To compliment the exhibition the team will also develop a self guided walk with a supporting audio piece detailing information on each tree.

After cross referencing our common interests with the general information on the Trinity Trees shared by David Hackett we have chosen a sample of ten different trees. See About/ the trees for a list of these trees with a brief outline of our reasons for choosing them. This sampling process will help us narrow our search to the final five or six trees that will form the basis for the research and resultant art works.

At this early stage of the project we haven’t a huge amount of information or imagery to share but as we initiate the sampling process in the next few weeks we will post some images of the samples.