Principles and Application of Fluorescence Microscopy

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Fluorescence is the luminescent emission
that results from absorption of photons. Fluorescence
is distinguished from its counterpart,
a longer-lasting afterglow called phosphorescence,
by the magnitude of the decay time.
Fluorescent emission ceases abruptly when the
exciting energy is shut off. The decay time, or
afterglow, of the emission is on the order of 10−8
sec and results in a negative frequency-shifted
emission. In contrast, the decay in phosphorescence
takes place in milliseconds to seconds.
The fluorescent effect is used in a number
of spectroscopy techniques, and it is particularly
useful for fluorescence microscopy. The
principal use of fluorescence microscopy is to
examine specimens that have been treated with
special fluorescent reagents. These reagents are
able to absorb light of a certain wavelength and
emit light at a longer wavelength slightly
shifted toward the red end of the spectrum from
the absorbed light. If, for example, blue light is
absorbed, green light will be emitted. Green is
shifted to yellow, yellow to red, and invisible
UV light to visible blue light. This phenomenon
is termed the Stokes shift and is defined as the
separation of the spectral maxima of excitation
and emission .
The λmax of the spectrum is typically ∼20 to
50 nm longer than that of the absorbed exciting
light. The Stokes shift, however, can range from
<10 to >100 nm ). Each fluorochrome
exhibits its own very specific absorption
and emission spectra, depending on the
structure of the molecules and sometimes also
on their surroundings.
Fluorescence microscopy allows selective
examination of a particular component of a
complex biomolecular assembly. A specimen
labeled with fluorescent dye(s) is illuminated
with filtered light of the absorbing wavelength
and is viewed using a barrier filter that is opaque
to the absorbing wavelength but which transmits
the longer wavelength of the emitted light.
The structures marked with the fluorescent
molecules will light up against the black background.
Additional experimental information
can be derived from the combination of optical
and biochemical responses exhibited by thefluorescent probe.
The growing importance in biology of fluorescence
microscopy is due to (1) the extraordinary
development of new fluorescent molecular
probes and (2) the development of improved
low-light-level imaging systems and
confocal microscopy techniques