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Fluorescence illumination and observation is the most rapidly
expanding microscopy technique employed today, both in the
medical and biological sciences, a fact which has spurred the
development of more sophisticated microscopes and numerous
fluorescence accessories. Epi-fluorescence, or incident light
fluorescence, has now become the method of choice in many
applications and comprises a large part of this tutorial. We
have divided the fluorescence section of the primer into several
categories to make it easier to organize and download. Please
follow the links below to navigate to points of interest.
Introductory Concepts - Fluorescence is a member of the
ubiquitous luminescence family of processes in which susceptible
molecules emit light from electronically excited states created
by either a physical (for example, absorption of light),
mechanical (friction), or chemical mechanism. Generation of
luminescence through excitation of a molecule by ultraviolet or
visible light photons is a phenomenon termed photoluminescence,
which is formally divided into two categories, fluorescence and
phosphorescence, depending upon the electronic configuration of
the excited state and the emission pathway. Fluorescence is the
property of some atoms and molecules to absorb light at a
particular wavelength and to subsequently emit light of longer
wavelength after a brief interval, termed the fluorescence
lifetime. The process of phosphorescence occurs in a manner
similar to fluorescence, but with a much longer excited state
lifetime.
Anatomy of the Fluorescence Microscope - In contrast to other
modes of optical microscopy that are based on macroscopic
specimen features, such as phase gradients, light absorption,
and birefringence, fluorescence microscopy is capable of imaging
the distribution of a single molecular species based solely on
the properties of fluorescence emission. Thus, using
fluorescence microscopy, the precise location of intracellular
components labeled with specific fluorophores can be monitored,
as well as their associated diffusion coefficients, transport
characteristics, and interactions with other biomolecules. In
addition, the dramatic response in fluorescence to localized
environmental variables enables the investigation of pH,
viscosity, refractive index, ionic concentrations, membrane
potential, and solvent polarity in living cells and tissues.
Additional information available at:
"http://micro.magnet.fsu.edu/primer/techniques/fluorescence/
fluorhome.html"
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