Electron Microscopy involves placing the sample in a vacuum and bombarding it with electrons to produce a final image. Special techniques must therefore be used in 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. The precise fixation procedure will depend not only on the tissue studied but also the type of ultrastructural information required. Glutaraldehyde is the most commonly used primary fixative. It penetrates rapidly and stabilises 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 60 oC.
|Sample Protocol for TEM||Tem Protocol for insects|
How does it work
The transmission electron microscope (TEM), in contrast to light microscopes, uses a beam of electrons in place of a beam of light. Electrons are negatively charged particles incapable of passing through glass. Therefore, the lenses of an electron microscope are electromagnets and by varying the strength of these lenses, the magnification of the image formed can be changed.
Electrons are charged particles, and because collision with charged 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 60,00 to 100,00 Volts. Because electron beams are invisible to the eye, the images they form are revealed on a fluorescent screen and can then be photographed.
The specimen must be extremely thin for the electrons to pass through it and create an image. Ultra thin sections are approximately 0.01um (100nm) thick, and are cut on an ultramicrotome. 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 ultrastructure.
The value of the electron microscope lies in its great resolving power. Transmission Electron Microscopes today are capable of resolving objects only 0.2 nanometers apart (One nanometre is a millionth of a millimetre) - just five times the diameter of a hydrogen atom, while in comparison the bright-field light microscope, has a resolution of approximately 0.2um and a useful magnification of X2,000.