Page 196 - Fundamentals of Light Microscopy and Electronic Imaging

PHYSICAL BASIS OF FLUORESCENCE 179

lites. While it is impractical to discuss specialized labeling techniques and methods of
fluorescence quantitation and analysis, it is important to note some of the principal
applications for which fluorescence microscopy is applied. These include:

• Determination of the intracellular distribution of macromolecules in formed struc-
tures such as membranes, cytoskeletal filaments, and chromatin. Fluorochrome-
conjugated metabolites, ligands, and proteins can be used to label membrane
channels and ion channels. Target molecules can also be labeled with fluorescent
antibodies (immunofluorescence microscopy) or with biotin or epitope tags, or
conjugated to fluorescent proteins such as green fluorescent protein (GFP) and
other agents. Multicolor labeling is possible, whereby several different molecular
species are labeled and viewed simultaneously using dyes that fluoresce at different
wavelengths.
• Study of intracellular dynamics of macromolecules associated with binding disso-
ciation processes and diffusion (fluorescence recovery after photobleaching, or
FRAP). FRAP techniques give the halftime for subunit turnover in a structure, bind-
ing constants, and diffusion coefficients.
• Study of protein nearest neighbors, interaction states, and reaction mechanisms by
fluorescence energy transfer or FRET and by fluorescence correlation microscopy.
In FRET, two different fluorochromes are employed, and the excitation of
the shorter-wavelength fluorochrome results in the fluorescence of the longer-
wavelength fluorochrome if the two moieties come within a molecular distance of
one another.
• Study of dynamics of single tagged molecules or molecular assemblies in vivo using
an extremely sensitive technique called total internal reflection fluorescence (TIRF)
microscopy.
• Determination of intracellular ion concentrations and changes in the concentra-

2

tions for several ionic species, including H ,Na ,K ,Cl ,Ca , and many other
metals. Ratiometric dyes are used, whose peak fluorescence emission wavelength
changes depending on whether the dye is in the free or bound state. The ratio of fluo-
rescence amplitudes gives the ion concentration.
• Organelle marking experiments using dyes that label specific organelles and
cytoskeletal proteins.
• Determination of the rates and extents of enzyme reactions using conjugates of fluo-
rochromes whose fluorescence changes due to enzymatic activity.
• Study of cell viability and the effects of factors that influence the rate of apoptosis
in cells using a combination of dyes that are permeant and impermeant to the
plasma membrane.
• Examination of cell functions such as endocytosis, exocytosis, signal transduction,
and the generation of transmembrane potentials using fluorescent dyes.

PHYSICAL BASIS OF FLUORESCENCE

Fluorescence is the emission of photons by atoms or molecules whose electrons are
transiently stimulated to a higher excitation state by radiant energy from an outside
source. It is a beautiful manifestation of the interaction of light with matter and forms