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PRINCIPLE:

Fluorescence microscopy is based on the principle of removal of incident illumination by selective absorption, whereas light that has been absorbed by the specimen and re-emitted at an altered wavelength is transmitted. The light source must produce a light beam of appropriate wavelength. An excitation filter removes wavelengths that are not effective in exciting the fluorochrome used. The light fluoresced by the specimen is transmitted through a filter that removes the incident wavelength from the beam of light. As a result, only light that has been produced by specimen fluorescence contributes to the intensity of the image being viewed.


DYES:


Acridine orange
Rhodamine — auramine
Calcofluor white
Fluorescein isothiocyanate
Lissamine rhodamine

USES:

1. For fluorochrome staining of tissues, cells or bacteria by using dyes like acridine orange, auramine O, calcofluor white, etc.

2. For immunofluorescence by using fluorescent dyes like fluorescein isothiocyanate & lissamine rhodamine to label the antibodies or antigens.