Fluorescence microscopy uses fluorescent dyes and ultraviolet light to study samples. When exposed to UV light, the dyes become excited and emit light of longer wavelengths. The microscope filters out the UV light and passes the emitted light through to view fluorescent specimens. Applications include using fluorescent dyes to tag and identify microbes, parasites, and antigens or antibodies in immunofluorescence techniques.

1. Fluorescence Microscope
Mr. Leyon Selvin Raj
Assistant Professor
Vivek Institute of Laboratory Medicine, Nagercoil

2. • Fluorescence microscopy is basically a method of
studying material which can be made to fluoresce,
either in its natural form or when treated with
chemicals capable of fluorescing.

3. Principle
• When fluorescence dyes are exposed to UV rays, they become
excited and convert this invisible short wavelength rays (UV) into
light of longer wavelength.

4. Working
1. The source of light may be a mercury lamp
which emits rays that pass through an
excitation filter.
2. The excitation filter allows only shorter
wavelength UV light, blocks others.
3. The exciting rays get reflected by a
dichromatic mirror which fall on the
specimen which is formerly stained by
fluorescent dye.
4. Then the specimen is focused under
microscope.

5. 5. The fluorescent dye absorbs the exciting
rays and emits fluorescent rays of
higher wavelength.
6. A barrier filter positioned after the
objective lens removes the UV light,
which could damage the viewer’s eyes.
And also the blue/violet light which
would reduce the contrast.

7. Applications
Epi-fluorescence microscope
• Simplest format of fluorescence microscope.
Auto fluorescence
• Cyclospora – Auto fluoresce
Microbes coated with fluorescent dye
• Acridine orange- malaria parasite-quantitative buffy coat examination.
• Auramine phenol- detection of tubercle bacilli
Immunofluorescence
• Florescent dye tagged antigens or antibodies.

8. THANK YOU