The Electron Microscope The electron microscope was an absolute essential development to further our scientific understanding and

to excel over the limitations of the light microscope such as the resolving power allowing scientists to see structures that previously were unknown due to their 'invisibility' under the light microscope. Comparison of the Electron microscope to the Light Microscope Light Microscope Light is the radiation source for a light microscope, this means it is the photon that is used for imaging. The resolution, meaning the ability to distinguish between two objects, is limited to half the wavelength of light, so is about 200nm depending on the size of the wave (400700nm). Electron Microscope (TEM) Electrons are used as the radiation source; they have a short wavelength of about 0.005nm and so are able to produce a resolution (ability to distinguish between two objects) , at best, of 0.1nm. These are generated by a hot tungsten filament.

The magnification of a light microscope is 1500x, meaning up until the electron microscope was introduced, many cell The magnification is much greater at organelles remained unknown. 250000x however, the specimen take much more time to prepare. A series of glass lenses are used to focus the light. The specimen does not need to be prepared as extensively as The fact that a vacuum needs to be the electron microscope however, dyes are occasionally created and a beam of electrons need to used to enhance the boundaries of specimens. be generated means a great deal of energy is needed compared to the light microscope. The main advantage of the light microscope over the electron is that images are produced in colour, which gives greater clarity in identifying different parts of the specimen. There are two types of electron microscopes; the first being a transmission electron microscope (TEM), introduced in the 1950s, followed by the scanning electron microscope (SEM). They are very different in the sense that the TEM allows electron pass through to the specimen, to be transmitted and the SEM simply scan the specimen. This difference changes the resolution, imaging process, size of specimens and preparation of specimens. Electromagnets provide a charge to focus the electron beam onto the specimen; they have to be used instead of glass lenses (as used in light microscopes) as glass does not affect the electron beam. The electrons are very sensitive to the electromagnets and therefore, changing the current of the electromagnets acts as a control for the electron beam. The electron beam is produced using a tungsten filament, similar to a light bulb filament. This filament is usually heated to around 2500 Kelvins, using 50,000 volts. This causes the ions in the metal filament to become unstable and as a result, electrons are thrown out of orbit it is these electrons that make up the beam.

The greater the voltage supplied, the greater the energy and therefore the shorter the wavelength of the electron. This is important because a smaller wavelength means a greater resolution. Recently, it has been possible to get a one million kilovolt electron microscope meaning the resolution can even distinguish individual cords on DNA This has recently been published by the Italian Institute of Technology, holding one of the world most powerful electron microscopes. It can be seen, as mentioned before that the individual cords of DNA are visible. The Transmission Electron Microscope Firstly, a beam of electrons is produced which are focused by a magnetic condenser lens on to the specimen. The specimen has to be extremely thin, around 100nm, however this can change slightly depending on the voltage used. The specimen is supported on a copper, gold or platinum mesh at 3mm in diameter. Some of the electrons are absorbed by the specimen, some are able to pass straight through. A magnetic objective lens directs the image onto a magnetic projector, which allow the electrons to hit photographic film. A fluorescent screen is used to help view the immediate image though the binoculars as electrons cannot be seen by humans due to the size of the wavelength. The image produced will be in black and white, with the darker areas showing denser areas of the specimen that have absorbed electrons and the lighter areas that have allowed electrons to pass through. The most common resolving power of a TEM is around 0.1 nm. The whole system must be kept in a vacuum to allow a clear path for electrons to reach the specimen otherwise, electrons would scatter and collide with the air particles. TEM Specimen Preparation

Firstly, preservatives are added to the specimens so they can be viewed as close to their living state as possible. Glutaraldehyde is used for preserving proteins and osmium tetroxide for lipids.

The specimen is then placed in a copper grid to support it under the electron microscope. To give a greater contrast in the final image, heavy metals such as gold, uranium or lead are used to stain the specimen.

The specimens are then dehydrated with organic solvents such as acetone or ethanol to prepare the specimens so they can be placed in a resin.

Once the specimen has been placed in an araldite resin, it is dried to form a solid block and then cut to slices 90nm thick using an ultra-microtome. This is so it is semi-transparent to electrons.

Scanning Electron Microscope As with the transmission electron microscope, the whole system is in a vacuum so electrons do not scatter and collide with air particles and the electron beam is generated using a tungsten filament. This beam is known as the primary beam. This beam is controlled by a series of magnetic lenses to ensure it hits the specimen; the magnetic lenses direct the beam to scan in a raster pattern. The electrons hit the surface of the specimen and cause the electrons on the surface to become excited and fly off. The beam of electrons that is produced as a result is known as the secondary beam. The secondary beam s and backscattered electrons are picked up by detectors around the specimen which build up an image using mapping software. The image produced will have a resolution of as small as 20nm. Specimen preparation for SEM `The first process is known as Fixation, it is the same as the TEM, as it is preserving the specimens so they will look the same as if they were alive. Finally, the specimen is coated in gold using a gold sputtering machine. It is these gold atoms on the coating that produce the secondary beam and cause fired electrons to backscatter helping to build an image, without it, the detector would receive a poor signal meaning the final image would be blurred and dark.

The part of the specimen that scientist want to view will need to be dissected, this can either be done manually be simply cutting away unwanted parts or chemically by dissolving tissue leaving behind the skeletal structure to be viewed.

an aluminium plate with a small stem is used to mount the specimen. The specimen is stuck onto the plate using silver conductive glue. The specimen is mounted to eliminate handling of the specimen which could cause artefacts - and to provide support.

The specimen is then dehydrated using either alcohol or acetone, this ensures the specimen will not collapse when mounted.

The specimens must then be cleaned to reduce artefacts ( objects that should not be there, usually from the specimen preparation). How this is done depends on the type of structure that is being viewed: Adding various chemicals can remove a waxy layer formed on substances and dissolve dirt A sonicator can be used which sends a high frequency though the specimen causing it to vibrate and shake off the dirt. The dirt/ dust particles can be picked off using ultra-fine pins.

Comparison of the TEM and SEM Scanning electron microscope Transmission electron microscope Lower resolution of 20nm High resolution, up to 0.1nm Both must be carried out in a vacuum to prevent electrons scattering Produces 3D Images, which can contain Only produces 2D images, which also artefacts contain artefacts Only scans the surface so thickness does Specimens must be extremely thin so not matter, specimen must be of a they are semi-transparent to electrons, reasonable size, preparation is much preparation is a much longer process, quicker, placed on an aluminium plate placed on a copper grid Lower magnification of 2x10^6, however Magnification of 50x10^6 better depth of field than the TEM $250,000- $750,000 $500,000 - $1,000,000

Resolution Conditions Imaging Specimens

Magnification Price