Fluorescence Microscopy

Fluorescence microscope is an optical microscope that uses fluorescence to generate an image.

Fluorescence microscopes are of two types:
1. Epifluorescence: it is called epifluorescence because the light source lies above the specimen and
objective lens
2. Confocal microscope: it uses optical sectioning to get better resolution of the fluorescent image

Principle of Fluorescence Microscope:

Instrumentation (Ray Diagram) fluorescence Microscope

The fluorescence microscope is based the principle of fluorescence. Fluorescence is a phenomenon in

which certain fluorescent chemicals absorb the light of particular wavelength and then emits the light of
longer wavelength with lesser content of energy.
The specimen is illuminated with light of specific wavelength which is absorbed by fluorophore
(fluorescent emitting compound) causing them to emit light of longer wavelength. The excited light is
separated from the emitted fluorescence through the use of spectral emission filter. The filters and
dichoric filter are selected to match the spectral ecitation and the emitted fluorescence of the
fluorophore, which is used to label the specimen. In this way the fluorescence of a single fluorophore is
detected in the form of image at a time. Multicolor image of several types of flurophores can also be
detected by using different combination of filters.

Instrumentation:
Light source of the epifluoresence microscope are Xenon arc, Mercury vapor, light emitting diodes
(LEDs), lasers etc. The excited light and the emitted fluorescence pass through the same light path (that
is through the objective lens). The excited light is focused on the specimen through the objective lens.
The fluorescence emitted from the specimen is focused to the detector by the same objective lens.