In transmission electron microscope (TEM) a beam of electrons is transmitted through the section of a specimen and an image is formed by the interaction of the electrons transmitted through the specimen. The image is magnified and focused onto an imaging device, such as a fluorescent screen or a photographic film, or to the computer screen.
TEM operates on the same basic principles as the light microscope but uses electrons instead of light, which makes it possible to get a resolution a thousand times better than with a light microscope. It was developed by Max Knoll and Ernst Ruska in Germany in 1931. Reinhold Rudenberg, the scientific director of Siemens, had patented the electron microscope in 1931, stimulated by family illness to make the poliomyelitis virus particle visible. Siemens produced the first commercial Transmission Electron Microscope (TEM) in 1939, but the first practical electron microscope had already been built at the University of Toronto in 1938, by Eli Franklin Burton, Cecil Hall, James Hillier and Albert Prebus.
By another analogy, a TEM works like a slide projector. A projector shines a beam of light through the slide and as the light passes through, it is affected by the object on the slide. This transmitted beam is then projected onto the viewing screen, forming an enlarged image of the slide.
Electron microscopes have much greater resolving power than light microscopes and use electromagnetic radiation that can obtain much higher magnifications of up to 2 million times, while the best light microscopes are limited to magnifications of 2000 times. Unlike Scanning Electron Microscope (SEM) that bounces electrons off the surface of a sample to produce an image, Transmission Electron Microscopes (TEM) shoots the electrons completely through the sample.
A source at the top of the microscope emits electrons that travel through vacuum in the column of the microscope. Instead of glass lenses focusing is done by electromagnetic lenses and the electrons are focussed into a very thin beam. The electron beam then travels through the specimen you want to study. Depending on the density of the material present, some electrons are scattered and disappear from the beam and unscattered electrons hit a fluorescent screen, which produces the image of the specimen with its different parts displayed in varied darkness according to their density. The image can be studied directly by the operator or photographed with a camera.
TEM WORKING
- The “Virtual Source” at the top represents the electron gun, producing a stream of monochromatic electrons. Electrons are charged particles, and because collision with molecules of air will absorb and deflect electrons and distort the beam, the optical system of an electron microscope must be evacuated of air. The electron source is produced by heating a tungsten filament at voltages usually ranging from 6,000 to 10,000 Volts. Because electron beams are invisible to the eye, the images they form are revealed on a fluorescent screen and can then be photographed.
- This stream is focused to a narrow beam by the use of condenser lenses. The first lens largely determines the “spot size”; the general size range of the final spot that strikes the sample. The second lens is controlled by the brightness knob and actually changes the size of the spot on the sample; changing it from a wide dispersed spot to a pinpoint beam.
- The beam is restricted by the condenser aperture, knocking out high angle electrons.
- The beam strikes the specimen and parts of it are transmitted. The specimen must be extremely thin for the electrons to pass through and create an image. Ultra thin sections are approximately 0.01um (100nm) thick, and are cut on an ultra microtome. Because ultra thin sections have little contrast, they must be stained with electron-absorbing heavy metal salts to provide contrast necessary to reveal details of the cells ultra structure.
- This transmitted portion is focused by the objective lens into an image.
- Optional Objective and selected area metal apertures can restrict the beam, the objective aperture enhancing contrast by blocking out high-angle diffracted electrons, the selected area aperture enabling the user to examine the periodic diffraction of electrons by ordered arrangements of atoms in the sample.
- The image is passed down the column through the intermediate and projector lenses and is enlarged all the way.
- The image strikes the phosphorescent image screen and light is generated, allowing the user to see the image. The darker areas of the image represent those areas of the sample that fewer electrons were transmitted through, meaning that they are thicker or denser. The lighter areas of the image represent those areas of the sample that more electrons were transmitted through, meaning that they are thinner or less dense. Fluorescent viewing screen is coated with a phosphor or scintillator material such as zinc sulphide.
SAMPLE PREPARATION
1. Fixation. The first step in sample preparation has the aim of preserving tissue in its original state. Fixatives must be buffered to match the pH and osmolarity of the living tissue. Glutaraldehyde is the most commonly used primary fixative. It penetrates rapidly and stabilizes proteins by forming cross links, but does not fix lipids. Osmium Tetroxide is used as a secondary fixative, reacting with lipids and acting as a stain. Following each fixation step, excess fixative must be washed out of the tissue.
2. Dehydration. Before sample can be transferred to resin all the water must be removed from the sample. This is carried out using a graded ethanol series.
3. Infiltration and Embedding in resin. The sample is infiltrated with a resin before being placed in an embedding mould, which is then polymerised in an oven at 60oC.
4. Sections of Embedded Material.Biological material contains large quantities of water. Since the TEM works in vacuum, the water must be removed. To avoid disruption as a result of the loss of water, you preserve the tissue with different fixatives. These cross-link molecules with each other and trap them together as stable structures. The tissue is then dehydrated in alcohol or acetone. After that, your specimen can be embedded in plastic that polymerize into a solid hard plastic block. The block is cut into thin sections by a diamond knife in an instrument called ultra microtome. Each section is only 50-100 nm thick. The thin sections of your sample is placed on a copper grid and stained with heavy metals. The slice of tissue can now be studied under the electron beam.
5. Negative Staining of Isolated Material.The isolated material that can be a solution with bacteria, is spread onto a support grid coated with plastic. A solution of heavy metal salt is added. The metal salt solution does not bind to the material but forms a “shadow” around it on the grid. The specimen will appear as a negative picture when viewing it in the TEM.