How and why we use the Scanning Electron Microscope (SEM)

Unlike optical microscopes, SEM magnification is not about the power of the objective lens.

Scanning Electron Microscope

Scanning Electron Microscope
Photographer: Martin Pueschel © Martin Pueschel

The SEM does have an objective lense - but theoretically it could work entirely without one. And that's why the SEM's function is not to 'image' the specimen, but to focus the beam to a spot.

An electron gun generates a beam with sufficiently small diameter. The magnification results from the ratio of the dimensions of the raster on the specimen and the raster on the display device. Magnification is controlled by the current (supplied to scan coils), or the voltage (supplied to deflector plates).

Specimens tend to charge when scanned by the electron beam. For conventional SEM imaging specimens must be electrically grounded to prevent scanning faults caused by electrostatic charge at the surface - therefore specimens get coated with an ultrathin coating, usually gold. This process is called sputter coating. This coating also increases the signal and surface resolution.

Viewing specimens in the SEM

The SEM has a monitor from which we as the operators view the specimen. The image is derived from the detection of excited electrons that are being bounced off the gold specimen at varying speeds and signals. The two detectors pick up the electron signals and via an analog/ digitising process the image is viewed on a monitor screen, captured and stored as a digital image (varying resolutions) on the computer’s hard drive.

Electron microscopes can only be viewed in black and white. This is because the electrons that are detected do not have a colour spectrum like that of the incandescent light in light microscopes. The only way to colour the derived SEM images is to computer generate pseudo colour images via imaging packages.

Specimen preparation and techniques

How it works with the High Vacuum Scanning Electron Microscope (SEM).

1. Fixation

Many specimens that come to the lab for scanning are often soft bodied and require a technique called fixation. The fixation technique is designed to preserve the cellular structure of the specimen so when observed under the SEM the dead specimen will (should) resemble that of a live specimen. This process offers the scientist a close up look of what the cells, ie skin, head, organs etc look like when alive. Various chemicals processes are used to fix the specimen. These chemicals are very dangerous to human health and care must be taken when dealing with them.

2. Extracting of the required part of the specimen

Often scientists are interested in only a part of the studied specimen, ie. the teeth of a snail, so our job in the SEM lab is to remove the wanted part from the specimen and prepare for SEM viewing. To extract the part we can use various ways including manual dissection (simply cut and pull the wanted part carefully away from the body) and chemical dissection (use chemicals which eat away the soft body material leaving only the wanted hard bodied sections such as the exoskeleton).

3. Cleaning

The specimens coming in from the field are usually dirty and often we can hardly make out what the specimen looks like. So if we are to view the specimen under high magnification it must be totally clean. There are three major ways for cleaning specimens:
a) Manual: this is where the dirt is picked off the specimen by very thin forceps and ultra fine pins. The dirt can also be removed by an eyelash, this is effective for delicate specimens.
b) Vibration: the grime can be removed by using a sonicator which sends a high vibration frequency through the specimen shaking off the dirt. This method is ideal for hard bodied specimens.
c) Chemical: various chemicals can be employed to actively remove the waxy layer on the surface of the specimens. Others simply dissolve or loosen the unwanted surface grime.

4. Drying

It is essential that the specimen is completely dry. The SEM works under a vacuum and for an image to be derived the specimen must be dry, if not the specimen will simply collapse or even better, blow up in front of your eyes in the vacuumed chamber. There are several ways to dry the specimens:
a) Air dried: many hard bodies specimens ie. insects are dried on capture so once cleaned they can be simply placed into the SEM
b) Critical Point dried: this complicated process involves simply, the replacement of liquid in the cells with gas. This process creates a completely dry specimen without or minimal cellular distortion.
c) Chemically dried: the wet specimen can be put through an alcohol dehydration series which replaces the water with alcohol and then the alcohol is slowly evaporated off leaving a dried specimen. Other dangerous chemicals can be employed to do the same liquid/air replacement and dehydration.

5. Mounting

Now that the chosen specimen has been fixed, cleaned and dried the next step is to mount the specimen on an aluminium stub. The stub is often a small, flat, round piece of metal that has a stem (looks like a flattened mushroom). The basic method of attachment is to glue the specimen or bits of the specimen to the stub which has been covered with double sided sticky tape and a thin layer of foil. The glue is a special silver conductive glue. We use this to ensure that the specimen (which is not conductive) will be grounded or earthed to the stub, this practice ensures that electron charging of the specimen in the SEM chamber is reduced.

The reasons for mounting the specimen include: stabilise the specimen in one place for viewing and manoeuvring in the SEM chamber; avoid the specimen from disappearing when being gold coated; and reduce the amount of handling of the specimen.

6. Gold Coating:

The gold splutter coater is a machine that we use to coat the mounted specimens in gold before they go into the SEM. The reason why we gold coat the specimens is because the SEM uses an electron beam instead of a light globe to illuminate the specimen. There are two detectors in the SEM chamber that are used to detect the two types of electrons that are bouncing of the gold metal specimen. It is these electrons - secondary and backscatter that go to make up an image of the specimen. This image of the electrons is what we are seeing, not the reflected light image we see in a light microscope. If the specimen is not finely covered with a metal like gold we will get a very poor signal thus the image derived will be very dark and perhaps not even there.

Mr Martin Pueschel , Scientific Illustrator
Sue Lindsay , Microscopy and Microanalysis Unit, Manager
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