Biology Reference
In-Depth Information
Exited state
Green light
(Lower energy)
Ground state
Blue light
(High energy)
FIGURE 9.28
Basic fluorescence. Light is absorbed by the fluorophore and a ground state electron is boosted to
an excited state (10
11
s). The electron then loses some of its energy by non-radiative relaxation (internal
conversion) to a lower energy excited state before returning to the ground state in the process called fluorescence
(~10
8
s). The emitted fluorescent light is of a longer wavelength (lower energy) than the light that was initially
absorbed. Courtesy of ScienceBlogs, LLC
Fluorescence and ESR are 'fast' techniques (i.e. they can be used to monitor fast processes).
Since NMR is greater than an order of magnitude slower, events that can be seen with fluo-
rescence and ESR may not be detectable by NMR. Detectable frequencies are ~10
5
sec
1
for
2
H'NMR and ~10
8
sec
1
for ESR and fluorescence.
The fluorescence technique most often used to investigate membrane structure, fluores-
cence polarization (FP), was first described by the French physicist Jean Baptiste Perrin in
1926 and is depicted in
Figure 9.29
. Perrin was a diverse and prolific physicist in the first
quarter of the 20
th
century, winning many awards including the 1926 Nobel Prize in
Physics. Plane-polarized light, produced by passing light through a polarizer, is used to
excite a fluorophore
[48
50]
. FP is based on the principle that if the fluorophore is immo-
bile, fluoresced light is emitted in the same plane as the plane-polarized excited light. If the
exciting light is polarized in the vertical direction, then the fluoresced light will also be
polarized in the vertical direction. This, however, is dependent upon a lack of movement
of the fluorophore during the excited state. If the fluorophore remains stationary from the
time it absorbs light until the time it emits light, the fluorescent light will remain
completely polarized. However, if the molecule rotates or tumbles during this time,
some of the light will be polarized in different directions (it becomes depolarized). The
extent of depolarization is monitored by comparing the intensity of vertically polarized
fluorescent light (observed through a vertical polarizer) to depolarized light (observed
e
