Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 217
Figure 6.18. Jablonski diagram for solvent relaxation.
relaxed state (R) will be observed. At intermediate temperatures,
where k S γ, emission and relaxation will occur
simultaneously. Under these conditions an intermediate
emission spectrum will be observed. Frequently this intermediate
spectrum (– – –) is broader on the wavelength scale
because of contributions from both the F and R states.
Time-dependent spectral relaxation is described in more
detail in Chapter 7.
Examples of temperature-dependent emission spectra
are shown in Figure 6.19 for the neutral tryptophan derivative
N-acetyl-L-tryptophanamide (NATA) in propylene glycol.
Solvents such as propylene glycol, ethylene glycol, and
glycerol are frequently used to study fluorescence at low
temperature. These solvents are chosen because their viscosity
increases gradually with decreasing temperature, and
they do not crystallize. Instead they form a clear highly vis-
Figure 6.19. Emission spectra of N-acetyl-L-tryptophanamide
(NATA) in propylene glycol. From [40].
cous glass in which the fluorophores are immobilized. The
presence of hydroxyl groups and alkyl chains makes these
compounds good solvents for most fluorophores.
As the temperature of NATA in propylene glycol is
decreased the emission spectrum shifts to shorter wavelengths
40 (Figure 6.19). This shift occurs because the decay
rate of fluorophores is not very dependent on temperature,
but the relaxation rate is strongly dependent on temperature.
Hence, at low temperature, emission is observed from the
unrelaxed F state. It is important to notice that the structured
1 L b (Chapter 16) emission of NATA is not seen even
at the lowest temperature (–68EC). This is because the
hydrogen bonding properties of propylene glycol persist at
low temperature. It is clear that the temperature-dependent
spectral shifts for NATA are due to the temperature dependence
of the orientation polarizability ∆f.
Another example of temperature-dependent spectra is
provided by Patman (Figure 6.20). 42 This fluorophore is a
lipid-like analogue of Prodan, which was developed to be a
probe highly sensitive to solvent polarity. 42 The basic idea
is that the amino and carbonyl groups serve as the electron
donor and acceptor, respectively. In the excited state one
expects a large dipole moment due to charge transfer (Figure
6.21), and thus a high sensitivity to solvent polarity. As
the temperature of propylene glycol is decreased, the emission
spectra of Patman shift dramatically to shorter wavelengths
(Figure 6.20). The effects of low temperature are
similar to those of low-polarity solvents. Because of the
decreased rate of solvent motion, emission occurs from the
unrelaxed state at low temperature.
6.5. PHASE TRANSITIONS IN MEMBRANES
Since its introduction as a solvent-sensitive probe, Prodan
and its derivative have become widely used to label biomolecules.
43–46 Alkyl or fatty acid chains have been added to
Prodan so that it localizes in membranes (Figure 6.22).
Acrylodan is a derivative of Prodan that can be used to label
sulfhydryl groups in proteins. Prodan is highly sensitive to
solvent polarity. Its emission maximum shifts from 410 nm
in cyclohexane to 520 nm in polar solvents (Figure 6.23). 47
Prodan and its derivatives have been especially useful
for studies of cell membranes and model membranes
because of its high sensitivity to the phase state of the membranes.
48–52 DPPC vesicles have a phase transition temperature
near 37E. When Prodan is bound to DPPC vesicles
the emission maximum shifts from 425 to 485 nm (Figure
6.24).