Principles of Fluorescence Spectroscopy

360 FLUORESCENCE ANISOTROPY
Figure 10.8. Excitation anisotropy spectrum of 9-anthroyloxy stearic
acid (9-AS) in propylene glycol at –52°C. The same excitation
anisotropy spectrum was observed for 2-AS, 7-AS, 12-AS and 16-AP.
Revised and reprinted with permission from [16]. Copyright © 1982,
American Chemical Society.
Figure 10.9. Excitation anisotropy spectrum of Green Fluorescent
Protein. Figure courtesy of Drs. Z. Gryczynski, L. Black and R. B.
Thompson.
Figure 10.9 shows excitation and emission anisotropy
spectra for GFP that was packaged into the heads of T4 bacteriophage.
17 Each phage head contains about 90 GFP molecules.
Given the size of the phage heads, the anisotropy is
expected to be close to the fundamental anisotropy, which
is near 0.38. Homotransfer between the concentrated GFP
molecules was suggested as the reason for the lower
Figure 10.10. Excitation anisotropy spectrum of indole in vitrified
propylene glycol. Also shown are the calculated absorption spectra of
the 1 L a and 1 L b states. Revised from [19].
anisotropy. The anisotropy is constant across the emission
spectrum, which is typical of emission anisotropy spectra.
10.3.1. Resolution of Electronic States from
Polarization Spectra
Some fluorophores can display complex anisotropy spectra,
even across the longest absorption band. One well-known
example is indole. 18–20 The anisotropy varies abruptly with
excitation wavelength across the long-wavelength absorption
band (Figure 10.10, top). This dependence was attributed
to the two excited states of indole ( 1 L a and 1 L b ), which
are responsible for the absorption from 250 to 300 nm. 21–22
The transition moments are thought to be at an angle of 90E
relative to one another. 23–24 Emission of indole occurs mainly
from the 1 L a state.
This complex anisotropy spectrum can be used to
determine the absorption spectra corresponding to the S 0 6
1 La and S 0 6 1 L b states. 19 This example illustrates the additivity
of anisotropies (eq. 10.6). This example is also impor-