Fluorescence microscopes use fluorescence to generate an image by exciting a specimen with one wavelength of light and detecting emitted light of a different, longer wavelength. Fluorescence microscopes have various applications including staining biological molecules, labeling proteins or other targets within cells using fluorescent antibodies in immunofluorescence techniques, and genetically modifying proteins to directly carry fluorescent protein reporters to track protein location.

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Fluorescent Microscopy
Fluorescence microscope refers to any microscope that uses fluorescence to generate
an image.
Fluorescent microscopes use an illumination method that is used to locate
fluorescently tagged material (protein, enzyme, genes, etc.) by exciting the specimen
with one wavelength of light in hopes that the fluorescence will appear by emitting a
light at a different wavelength. A fluorescence microscope is an optical microscope
that uses fluorescence and phosphorescence instead of, or in addition to, reflection
and absorption to study properties of organic or inorganic substances. Working of
Fluorescence microscope is as follows:
 The specimen is illuminated with light of a specific wavelength (or wavelengths)
which is absorbed by the fluorophores.
 These fluorophores 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.
Figure: Working principle of Fluorescence Microscope.
APPLICATIONS OF FLUORESCENT MICROSCOPES

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The following are some of the applications of fluorescent microscopes:
Biological Fluorescent Stains: Many fluorescent stains have been designed for a range
of biological molecules. Some of these are small molecules that are intrinsically
fluorescent and bind a biological molecule of interest. Many nucleic acid (fluorescent)
stains bind the minor groove of DNA, thus labeling the nuclei of cells.
Fluorophores / Fluorochromes: These are many fluorescent molecules that can be
chemically linked to a different molecule which binds the target of interest within the
sample.
Immunofluorescence: Immunofluorescence is a technique that uses the highly specific
binding of an antibody to its antigen to label specific proteins or other molecules within
the cell. A sample is treated with a primary antibody specific for the molecule of
interest. A fluorophore can be directly conjugated to the primary antibody.
Alternatively, a secondary antibody, conjugated to a fluorophore, which binds
specifically to the first antibody can be used. For example, a primary antibody raised
in a mouse which recognizes tubulin combined with a secondary anti-mouse antibody
derivatized with a fluorophore could be used to label microtubules in a cell.
Figure: Basic mechanism of immunofluorescence
Primary immunofluorescence involves an antibody with a fluorophore group bound to
it directly binding to the epitope of the antigen for which it is specific. Once the antibody

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binds to the epitope, the sample can be viewed under a fluorescent microscope to
confirm the presence of the antigen in the sample. Secondary immunofluorescence
involves an untagged primary antibody that binds to the epitope of the antigen.
However, after the primary antibodies have bound to their targets, a secondary
antibody (tagged with a fluorophore) comes along which binds to the primary antibody.
Fluorescent Proteins: The modern understanding of genetics and the techniques
available for modifying DNA allow scientists to genetically modify proteins to also carry
a fluorescent protein reporter. In biological samples this allows a scientist to directly
make a protein of interest fluorescent. The protein location can then be directly
tracked, including in live cells.