Preparation of specimens for viewing in the SEM
Contents
1. Fixation
2. Extracting the required part of the specimen
3. Cleaning
4. Drying
5. Mounting
6. Gold coating
1. Fixation
Many specimens that come to the lab for scanning are soft bodied and require a technique called fixation. The fixation technique is designed to preserve the cellular structure, so that the scientist can view the specimen in a form as close as possible to that of a living animal. Various chemical 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 the required part of the specimen
Often scientists are interested in only a part of the studied specimen, eg. the teeth of a snail, so our job in the SEM lab is to remove the wanted part from the specimen and prepare it for SEM viewing. There are various methods we can use to extract that part. One way is to simply cut and pull the part carefully away from the body (a manual dissection). Another way is to use chemicals which eat away the soft body material leaving only the required hard bodied sections (the exoskeleton, the snails teeth).
3. Cleaning
The specimens coming in from the field are usually dirty and often it is difficult to know what the original animal looked like. If we are to view the specimen under high magnification it must be totally clean. There are three major ways for cleaning specimens:
- 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.
- Vibration: the grime can be removed by using a sonicator which sends a high frequency vibration through the specimen shaking off the dirt. This method is ideal for hard-bodied specimens.
- Chemical: Various chemicals can be employed to chemically remove the waxy layer on the surface of the specimen. 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 blow up in the vacuumed chamber. There are several ways to dry the specimens;
- Air-dried: many hard bodies specimens, for example insects, are dried on capture so once cleaned they can be simply placed into the SEM
- Critical Point Drying: this complicated process involves simply the replacement of liquid in the cells with gas. This process creates a completely dry specimen with minimal or no cellular distortion.
- Chemical dehydration: 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 - it looks a bit 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, thus ensuring that electron charging of the specimen in the SEM chamber is reduced.
The reason for mounting the specimen is to:
- stabilise the specimen in one place for viewing and manoeuvring in the SEM chamber
- avoid the specimen disappearing when being gold coated
- reduce the amount of handling of the specimen
6. Gold coating
The gold sputter coater is a machine that we use to coat the mounted specimens in gold before they go into the SEM. The specimens must be gold coated because most material (but not gold) is tranparent to the electron beam used by the SEM. There are two detectors in the SEM chamber which create a signal from electrons bouncing of the gold-coated specimen. These are used to make up an image of the specimen. If the specimen is not finely covered with an electron-opaque substance like gold, the electron beam would travel right through the specimen, creating no image and probably destroying the specimen too!
Copyright © 2003, Australian Museum
