School of Life Sciences

The Sussex Centre for Advanced Microscopy

Why do we use Electron Microscopes?

General

	You are probably familiar with light microscopes, but what are electron microscopes, why were they developed and why are they so widely used?
	The answer is all about resolution (the ability to see fine detail in the image). Resolution is dependent (among other factors) upon the wavelength of the illumination used in the microscope.

The Light Microscope uses light as the illuminating source. The highest effective magnification of a light microscope is about 1,000X; at magnifications beyond this no further detail in the image is acquired. The best possible theoretical resolution of a light microscope is about 0.25 microns.

The electron microscope (or EM), however, has a beam of electrons as its source of illumination. As the wavelength of this electron beam is shorter than that of light then resolution is improved. In 'transmission' EMs (see below) this is typically around a few tenths of a nanometre (i.e. 0.0002 microns).

Because of this improved resolution the image in an electron microscope may be magnified up to around 500,000X and we can see much finer detail in the specimen.

Types of Electron Microscope

There are two basic types of EM: the Transmission EM (TEM) and the Scanning EM (SEM).

In the TEM the electron beam passes through the specimen, which is routinely a thin section or a particulate sample (e.g. proteins or virus particles).

The TEM is used to observe the fine internal structure (ultrastructure) of the specimen (i.e. all the cellular organelles).

In the SEM the electron beam is scanned over the specimen, which is usually what we call a ‘bulk’ sample (e.g. a whole insect, plant part, etc.).

The SEM is therefore utilised to discern the external appearance (or surface morphology) of the specimen.

Both types of EM operate under a high vacuum as the electron beam has limited energy (gas or water molecules would hinder the passage of the electrons).

The Scanning Electron Microscope (SEM)

SEM Preparation

The specimen is firstly fixed (i.e. preserved in as close to its living state as possible).

Specimens are then dehydrated (i.e. their water is removed and replaced with ethanol [alcohol]). This is required because electron microscopes operate under a high vacuum and therefore no water should be allowed into the microscopes.

Next, the specimens are ‘critical-point dried’. This is carried out in a specialised pressure vessel (a critical-point dryer) and involves replacing the ethanol with liquid carbon dioxide (CO2). The liquid CO2 is then ‘sublimed’ (converted from liquid to gas) instantaneously by heating.

The critical-point drying produces a perfectly dry but turgid (i.e. not collapsed or shrivelled) specimen.

The specimen is then stuck onto a special SEM sample holder (called a stub) and then ‘sputter-coated’ with a very thin layer of gold in a coating device. This gives the sample a conductive layer and prevents charging in the SEM.

The specimen is now ready for viewing in the SEM.

What do we use SEMs for?

The SEM is used for looking at the surface features (external morphology) of a specimen in fine detail.

Some examples of specimens commonly looked at in our lab are: Drosophila (fruitflies), feather mites, the cochlea (part of the inner ear) and floral parts.

The SEM may also be used to find out which elements are present within a specimen. This is carried out with an elemental analysis detector, which can collect the x-rays given off by a specimen when the electron beam hits it.

The useful feature of these x-rays is that each individual element within a sample gives off x-rays of a particular type. Therefore, if we look at the types of x-rays collected, we can tell which elements have emitted them.

The Transmission Electron Microscope (TEM)

TEM Preparation

The specimen is firstly fixed (i.e. preserved in as close to its living state as possible).

Specimens are then dehydrated (i.e. their water is removed and replaced with ethanol [alcohol]). This is required because electron microscopes operate under a high vacuum and therefore no water should be allowed into the microscopes.

The specimens are then placed in a liquid resin (similar to an araldite-type glue).

After a few days (and several resin changes) the specimens are heat-polymerised (the resin is hardened) to produce a firm specimen block.

Very thin sections are then cut on a special section-cutter called an ultramicrotome.

The sections are then stained in solutions of heavy metals (usually uranium and lead) which produce contrast in the image by causing scattering (diversion) of the electron beam.

What do we use TEMs for?

The TEM is used for looking at the fine detail of structure within the specimen.

Within cells this fine detail is called ultrastructure.

Being able to see the ultrastructure within cells means that we can see all the cellular organelles in detail.

We can see changes in the ultrastructure within cells (e.g. after a drug treatment) by a method called morphometric analysis.

Morphometric analysis is carried out by taking images with a digital camera. The digital images are then analysed on a computer and the changes in relative amounts of the organelles can be measured.

The TEM may also be used to see where specific proteins are within a cell. This is done by a method known as immunogold labelling.