Fluorescence microscopy is a powerful technique used in many fields of science, especially medical sciences. It works by exciting fluorescent molecules in a specimen with one wavelength of light, which causes them to emit a different wavelength of light. A fluorescence microscope uses filters to isolate the emitted light and form an image with high contrast against a dark background. This allows very sensitive observations and measurements by detecting the natural or dye-induced fluorescence of cells and molecules.

1.  The absorption and subsequent re-radiation of light by organic and inorganic
specimens is typically the result of well-established physical phenomena
described as being either fluorescence or phosphorescence. The emission of
light through the fluorescence process is nearly simultaneous with the
absorption of the excitation light due to a relatively short time delay between
photon absorption and emission, ranging usually less than a microsecond in
duration. When emission persists longer after the excitation light has been
extinguished, the phenomenon is referred to as phosphorescence.
British scientist Sir George G. Stokes first described fluorescence in 1852 and was
responsible for coining the term when he observed that the mineral fluorspar
emitted red light when it was illuminated by ultraviolet excitation. Stokes noted that
fluorescence emission always occurred at a longer wavelength than that of the
excitation light. Early investigations in the 19th century showed that many specimens
(including minerals, crystals, resins, crude drugs, butter, chlorophyll, vitamins, and
inorganic compounds) fluoresce when irradiated with ultraviolet light. However, it
was not until the 1930s that the use of fluorochromes was initiated in biological
investigations to stain tissue components, bacteria, and other pathogens. Several of
these stains were highly specific and stimulated the development of the fluorescence
microscopeThe technique of fluorescence microscopy has become an essential tool
in biology and the biomedical sciences, as well as in materials science due to
attributes that are not readily available in other contrast modes with traditional
optical microscope

3.  Fluorescence microscopy is a very powerful technology
that can be used in many fields of science, particularly
medical sciences. This technology is based on the
natural phenomena of fluorescence, which is the
absorption and release of light by organisms. A
fluorescence microscope is able to detect this emission
of light from an organism. If an organism or sample
does not naturally have fluorescence, various dyes and
even genes can be inserted into their genome such that
they can emit light. Due to the nature of the organism
emitting light, images are seen against a dark
background, allowing for very specific and sensitive
measurements and observations.

5. Principle of Fluorescence
 The specimen is illuminated with light of a specific wavelength (or
wavelengths) which is absorbed by the fluorophores, causing them
to emit light of longer wavelengths (i.e., of a different color than the
absorbed light). The illumination light is separated from the much
weaker emitted fluorescence through the use of a spectral emission
filter. Typical components of a fluorescence microscope are a light
source (xenon arc lamp or mercury-vapor lamp), the excitation
filter, the dichroic mirror (or dichroic beamsplitter), and theemission
filter (see figure below). The filters and the dichroic are chosen to
match the spectral excitation and emission characteristics of the
fluorophore used to label the specimen.[1] In this manner, the
distribution of a single fluorophore (color) is imaged at a time.
Multi-color images of several types of fluorophores must be
composed by combining several single-color images.[1]
 Most fluorescence microscopes in use are epifluorescence
microscopes (i.e., excitation and observation of the fluorescence are
from above (epi–) the specimen). These microscopes have become
an important part in the field of biology, opening the doors for more
advanced microscope designs, such as the confocal microscope and
the total internal reflection fluorescence microscope (TIRF).

6.  A. fluorescence micsoscopy can be used with any light microscope
B. fluorescence micsoscopy can be used with live specimens
C. fluorescence micsoscopy can be used with an electron microscope
D. fluorescence micsoscopy is less expensive and easier to use
DISADVANTAGES:-
Photobleaching and also some limitations arise from
1) the availability of target specific antibodies. If they are not
commercially available you have to make them which is time
and money consuming.
2) Specificity of the antibody. Sometimes the antibody will
bind other targets not intended for visualization and make it
difficult to locate the target.
3) Ability of the antibody to diffuse to the target. Sometimes
the target protein it sequestered in a location that is difficult
to reach with the antibody.