Biology Reference
In-Depth Information
FIGURE 9.27 George Gabriel Stokes (1819 e 1903) [58] .
observation, that the most powerful and versatile technique in modern membrane studies,
fluorescence, had its humble beginnings.
The technique of fluorescence is reviewed in two comprehensive topics by Joseph Lako-
witz [48,49] . Fluorescence occurs after a molecule (a fluorophore) absorbs light (is excited)
at one wavelength and radiates (emits) light at a longer wavelength. Basic fluorescence is
depicted in Figure 9.28 . The specific frequency of excitation and emission are dependent
on the characteristics of the fluorophore. The unexcited molecule has its electrons in the
lowest energy or ground state, S 0 . Upon absorbing light, a ground state electron is boosted
to its first (electronically) excited state, S 1 . A fluorophore in its excited S 1 state can relax by
two basic pathways. It can undergo non-radiative relaxation (internal conversion) by losing
heat to its surroundings, before returning to the ground state (S 1 /
S 0 ) in a process referred
to as fluorescence. The electron spin direction does not change during fluorescence. However,
if the electron spin changes during the excited state, a triplet state, T, results. Emitted radia-
tion during return to the ground state (T
S 0 ) is called phosphorescence. Since most
membrane studies involve fluorescence, phosphorescence will not be discussed, and is not
included in Figure 9.28 .
The time domains involved in fluorescence are very fast. The process of light absorption
and production of the excited state (S 0 /
/
S 1 ) takes ~10 11 sec. It takes ~10 8 sec to return
from the excited to the ground state (S 1 /
S 0 ). The average time in which the fluorophore
stays in its excited state before emitting a photon is referred to as the fluorescence lifetime.
It is the time between these two events, light absorbance and fluorescence, that is important
for membrane structure studies. In contrast, phosphorescence is relatively slow (
10 3 sec).
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