The Essential Components of Fluorescence microscopes are as follows:
The principles of magnification and resolution of a fluorescence microscope are no different from a bright field microscope. The essential components are light source, heat filter, exciter filter. Condenser and barrier filters.
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1. Light source:
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This is provided by a bright mercury vapor arc lamp. As against the normal incandescent bulb, this lamp provides shorter wave length light rays such as UV, violet and blue.
The lamp always comes with a transformer as sometimes a voltage of 18, 0000 volts are required.
Mercury vapor lamps produce light rays in range of200 – 400nm (UV) and visible rays in the range of above 780nm.
Mercury vapor lamps are not only expensive but also harmful. Hence several precautions are necessary. The bulbs are pressurized hence there is danger of their explosion. Eyes should not be directly exposed to the bulb as the rays are harmful.
2. Heat filter:
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Infrared rays produced by the lamp generate considerable heat, besides they are of no use in fluorescence.
In order to eliminate these rays a heat filter is placed in front of the lamp and before the condenser to absorb heat. Heat filter however does not prevent the transmission of UV and the visible rays.
3. Exciter filters:
The light cooled down by the heat filter next passes through the exciter filter which absorbs all but shorter waves that are needed to excite the flurochrome dye coated specimen on the slide. These filters which are dark allow only green, blue, violet or UV rays.
4. Condenser:
For best results always a dark field condenser is used, because in a dark background even mild fluorescence can be easily detected-Anolher advantage of this condenser is that it deflects majority of the UV rays thus protecting the observer’s eyes. In order to achieve this NA of the objective is always kept at 0.05 less than that of the condenser.
5. Barrier filter:
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This is situated in the body tube of the microscope between the objectives and the eye piece to remove all remnants of the exciting light so
That only the fluorescence is seen. When excitation is by UV, the exciter filters are dark and barrier filters will be almost colorless. On the other hand, if blue excitation is used, the barrier filters are either yellow or deep orange in color.