Principles of Fluorescence Spectroscopy

408 TIME-DEPENDENT ANISOTROPY DECAYS
glycerol as compared to ethylene glycol. The lower panel
shows histograms of the occurrence of each correlation
time in the examples. While the distribution is wide in glycerol,
the results demonstrate the possibility of imaging
using correlation times. One can imagine such results being
extended to intracellular fluorophores to obtain contrast
based on the mobility of fluorophores in each region of a
cell.
11.10. MICROSECOND ANISOTROPY DECAYS
11.10.1. Phosphorescence Anisotropy Decays
The information available from an anisotropy decay is limited
to times during which emission occurs. For this reason
it is usually difficult to obtain reliable data at times longer
than three intensity decay times. Even for probes having
relatively long lifetimes such as dansyl-lysine (Section
11.5.4), this time window is too small to effectively study
membrane-bound proteins.
Rotational motions at longer times can be measured
using phosphorescence anisotropy decays. 106–112 These
experiments are illustrated by studies of the sarcoplasmic
reticulum Ca 2+ -ATPase, which is a 110-kD transmembrane
protein. Not many phosphorescence probes are available,
and one of the most commonly used probes is erythrosin
(Figure 11.35), which in deoxygenated solution displays a
phosphorescence decay time near 100 µs. Typical phosphorescence
anisotropy decays are shown for erythrosinlabeled
Ca 2+ -ATPase. In this case the ATPase was aggregated
in the membrane by melittin. The extent of aggregation
Figure 11.35. Phosphorescence decays of erythrosin-labeled Ca 2+ -
ATPase in sarcoplasmic reticulum vesicles. Anisotropy decays were
obtained in the absence of melittin (A), in the presence of acetylated
melittin (B), or native melittin (C). Revised and reprinted with permission
from [110]. Copyright © 1995, American Chemical Society.
was greater for native melittin than for acetylated melittin,
which neutralizes the positive charges on melittin and
decreases its interactions with the Ca 2+ -ATPase. Because
the extent of crosslinking is less for acetylated melittin, so
that the anisotropy decays more rapidly. There are several
disadvantages to the use of erythrosin, eosin and other
phosphorescent probes. The signals are usually weak due to
the low phosphorescence quantum yields. Rigorous exclusion
of oxygen is needed to prevent quenching. And, finally,
fundamental anisotropies are usually low, near 0.1,
resulting in decreased resolution of the anisotropy decays.
11.10.2. Long-Lifetime Metal–Ligand Complexes
Another approach to measuring long correlation times is to
use luminescent metal–ligand complexes (MLCs). These
probes display lifetimes ranging from 100 ns to 10 µs 113–116
(Chapter 20). These probes are typically complexes of transition
metals with diimine ligands. A lipid MLC probe was
made by covalently linking two phosphatidylethanolamine
(PE) lipids to an Ru-MLC that contained two carboxyl
groups. The MLC-lipid probe was then incorporated into
DPPG vesicles (Figure 11.36). The maximum in the differential
phase angle (∆ ω ) is near 1 MHz, suggesting slow rotational
diffusion. The lifetime of the Ru-PE 2 probe was near
Figure 11.36. Frequency-domain anisotropy decays of Ru(bpy) 2
(dcbpy)–PE 2 in DPPG vesicles. Reprinted from [114], with permission
from Academic Press, Inc.