Electron microscopy (EM) is a technique for obtaining high-resolution images of biological and non-biological specimens. It is used to investigate the detailed structure of tissues, cells, organelles, and macromolecular complexes. The high resolution of EM images results from the use of electrons as the source of illuminating radiation.
There are two main types of electron microscope – the transmission EM (TEM) and the scanning EM (SEM)
Scanning Electron Microscopy:
type of electron microscopy designed for directly studying the surfaces of solid objects, that utilizes a beam of focused electrons of relatively low energy as an electron probe that is scanned in a regular manner over the specimen.
SEM images of surfaces appear to be three-dimensional (3D) but there is no measurable depth information in the image.
So How Does It Work?
A scanning electron microscope scans a beam of electrons over a specimen to produce a magnified image of an object:
1. Electrons are fired into the machine.
2. The main part of the machine (where the object is scanned) is contained within a sealed vacuum chamber because precise electron beams can't travel effectively through air.
3. A positively charged electrode (anode) attracts the electrons and accelerates them into an energetic beam.
4. An electromagnetic coil brings the electron beam to a very precise focus, much like a lens.
5. Another coil, lower down, steers the electron beam from side to side.
6. The beam systematically scans across the object being viewed.
7. Electrons from the beam hit the surface of the object and bounce off it.
8. A detector registers these scattered electrons and turns them into a picture.
9. A hugely magnified image of the object is displayed on a TV screen.
Transmission Electron Microscopy
Transmission electron microscopy (TEM) is a technique used to observe the features of very small specimens. The technology uses an accelerated beam of electrons, which passes through a very thin specimen to enable a scientist the observe features such as structure and morphology.
TEM produces 2D high-resolution, black and white image from the interaction that takes place between prepared samples and energetic electrons in the vacuum chamber.
So How Does It Work?
A transmission electron microscope fires a beam of electrons through a specimen to produce a magnified image of an object.
1. A high-voltage electricity supply powers the cathode.
2.The cathode is a heated filament, a bit like the electron gun in an old-fashioned cathode-ray tube (CRT) TV. It generates a beam of electrons that works in an analogous way to the beam of light in an optical microscope.
3.An electromagnetic coil (the first lens) concentrates the electrons into a more powerful beam.
4.Another electromagnetic coil (the second lens) focuses the beam onto a certain part of the specimen.
5.The specimen sits on a copper grid in the middle of the main microscope tube. The beam passes through the specimen and "picks up" an image of it.
6.The projector lens (the third lens) magnifies the image.
7.The image becomes visible when the electron beam hits a fluorescent screen at the base of the machine. This is analogous to the phosphor screen at the front of an old-fashioned TV .
8.The image can be viewed directly (through a viewing portal), through binoculars at the side, or on a TV monitor attached to an image intensifier (which makes weak images easier to see).
Image of How it Works:
So Why Use SEM and TEM Over Light Microscopy?
SEM and TEM provide different perspectives into cellular structure. TEM offers valuable information on the inner structure of the sample, such as crystal structure, morphology and stress state information, while SEM provides information on the sample's surface and its composition. Electron microscopes, such as SEM and TEM have a greater depth of field compared to light microscopes. Their higher resolution gives the human eye the subjective impression of a higher depth of field
The Limitations of SEM and TEM:
SEM and TEM are very large and expensive to navigate. Due to the complexity of the item, special training is required not only to operate the product, but also to be able to accurately analyse the data that the sample imaging provides.
The sample must be able to stand the vacuum chamber: this requires the sample to be sliced thin enough for electrons to pass through.
Another limitation is the inability to analyze live samples. This means that biological interactions of specimens cannot be properly observed