Principles of Fluorescence Spectroscopy

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Time-resolved measurements are widely used in fluorescence
spectroscopy, particularly for studies of biological
macromolecules and increasingly for cellular imaging.
Time-resolved measurements contain more information
than is available from the steady-state data. For instance,
consider a protein that contains two tryptophan residues,
each with a distinct lifetime. Because of spectral overlap of
the absorption and emission, it is not usually possible to
resolve the emission from the two residues from the steadystate
data.
However, the time-resolved data may reveal two decay
times, which can be used to resolve the emission spectra
and relative intensities of the two tryptophan residues. The
time-resolved measurements can reveal how each of the
tryptophan residues in the protein is affected by the interactions
with its substrate or other macromolecules. Is one of
the tryptophan residues close to the binding site? Is a tryptophan
residue in a distal domain affected by substrate
binding to another domain? Such questions can be
answered if one measures the decay times associated with
each of the tryptophan residues.
There are many other examples where the timeresolved
data provide information not available from the
steady-state data. One can distinguish static and dynamic
quenching using lifetime measurements. Formation of static
ground-state complexes do not decrease the decay time
of the uncomplexed fluorophores because only the unquenched
fluorophores are observed. Dynamic quenching
is a rate process acting on the entire excited-state population,
and thus decreases the mean decay time of the entire
excited-state population. Resonance energy transfer is also
best studied using time-resolved measurements. Suppose a
protein contains a donor and acceptor, and the steady-state
measurements indicate the donor is 50% quenched by the
acceptor. The result of 50% donor quenching can be due to
100% quenching for half of the donors, or 50% quenching
Time-Domain
Lifetime
Measurements
of all the donors, or some combination of these two limiting
possibilities. The steady-state data cannot distinguish
between these extreme cases. In contrast, very different
donor intensity decays would be observed for each case. If
all the donors are 50% quenched by the acceptors, and the
acceptors are at a single distance, then the donor decay will
be a single exponential with a lifetime of half the
unquenched lifetime. If 50% of the donors are completely
quenched and 50% are not quenched, then the donor lifetime
will be the same as the unquenched lifetime. A multiexponential
decay would be observed if the donor is partially
quenched by the acceptor and some of the donors do not
have a nearby acceptor. The time-resolved donor decays are
highly informative about the purity of the sample as well as
the donor-to-acceptor distance.
There are many other instances where lifetime measurements
are advantageous over steady-state measurements.
One important application is cellular imaging using
fluorescence microscopy. When labeled cells are observed
in a fluorescence microscope, the local concentration of the
probe in each part of the cell is not known. Additionally, the
probe concentration can change during the measurement
due to washout or photobleaching. As a result it is difficult
to make quantitative use of the local intensities. In contrast,
if the probe emission is well above the background signal,
fluorescence lifetimes are typically independent of the
probe concentration. Many fluorescence sensors such as the
calcium probes display changes in lifetime in response to
analytes. Also, resonance energy transfer (RET) reveals the
proximity of donors and acceptors by changes in the donor
lifetime. Because of advances in technology for timeresolved
measurements, it is now possible to create lifetime
images, where the image contrast is based on the lifetime in
each region of the sample. Fluorescence lifetime imaging
microscopy, or FLIM, has now become an accessible and
increasingly used tool in cell biology (Chapter 22). An
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