Fluorescence microscope involves staining of the specimens with special fluorescent dyes and is an indispensible instrument in diagnostic microbiological laboratories
A fluorescence microscope differs from an ordinary light microscope in the following respects.
(i) Light source is a mercury vapor lamp
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(ii) A dark field condenser is used instead of the normal Abbe condenser
(iii) Three sets of filters are employed to alter the light rays that pass through the instrument and reach the eye.
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The Principle of Fluorescence Microscope:
Light exists as a form of energy and is transmitted in the form of waves. When a wave of light encounters any obstruction like when it reaches molecules of a substances the electrons of the molecule at the outer orbit get excited and oscillate in resonance with the light waves.
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When a substance absorbs light it essentially absorbs energy. This energy that is absorbed cannot just disappear.
It should be transmitted out in some form or it should be converted to some other form of energy as it happens in photosynthesis.
When light is transmitted by the energized (excited) molecules it is said to be Photoluminescence. In photoluminescence, the light absorbed is not transmitted immediately. There is always a time lapse between absorption and emission.
If the time lapse is greater than 10(1/10,000) seconds it is said to be Photoluminescence. If on the other hand the time lapse is less than 10 seconds it is known as fluorescence.
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When a fluorescent dye is applied to a molecule it absorbs a wave length of light (excitation wave length) and emits a light ray of a different wave length (Emission wave length).
In other words, the wave length of light changes as it emerges out of a fluorescent compound. Usually the emission light will have a longer wavelength.
For instance, when the fluorescent dye fluoresce in isothiocyanate is illuminated with blue light, it emits green light.
This is due to the fact that light rays of shorter wavelength have more energy than those of longer wavelength and emission light always has less energy.
Microbiological specimens to be observed under a fluorescence microscope are coated with fluorescent componds (flourochromes) such as Auramine O, Acridine orange, Fluoroscein, etc.