Principles of Fluorescence Microscope and Fluorescent Dyes

PRINCIPLE OF FLUORESCENCE MICROSCOPE:

Fluorescence microscope 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.

Tubercle bacilli with fluorescent dye

DYES USED IN FLUORESCENCE MICROSCOPE:

• Acridine orange
• Rhodamine-auramine
• Calcofluor white
• Fluorescein isothiocyanate
• Lissamine rhodamine

USES OF FLUORESCENCE MICROSCOPE:

1. For 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.