Fluorolog-3 v.2.2 (10 Sep 2002)
Introduction to Lifetime Measurements
8-4
Types of lifetime scans
The type of scan defines which measurement will be acquired. In lifetime operation,
four scan types are available:
•
Lifetime
•
Lifetime-resolved
•
Dynamic depolarization
•
Time-resolved.
Recalling an experiment allows the user to retrieve the experimental parameters. Each
scan type is defined below.
Lifetime acquisition
The
lifetime acquisition
type of scan determines accurate lifetimes from simple single-
component systems as well as complex heterogeneous systems. The measurement re-
cords the phase shift and modulation at specified frequencies for an unknown sample
relative to a reference standard.
Lifetime-resolved acquisition
A
lifetime-resolved acquisition
scan resolves up to three components of overlapping
spectra based on differences in the fluorescence lifetimes. More complex systems can
result in improved resolution of one or more spectra but complete resolution requires
additional manipulation of data acquisition parameters such as excitation wavelength.
An application using lifetime-resolved-acquisition scans can spectrally resolve tyrosine
and tryptophan emission spectra from a protein containing both residues. To improve
resolution, simply measure the lifetime and obtain the spectral characteristics of the in-
dividual components prior to conducting a lifetime-resolved acquisition scan. Gener-
ally, this technique works best if a factor of at least 1.5 exists between the lifetimes be-
ing resolved spectrally.
Anisotropy-decay acquisition
An
anisotropy-decay acquisition
experiment choice allows the study of rotational prop-
erties of fluorescent molecules and probes. As the fluorophore rotates, a change in the
polarization occurs. Monitoring this change provides information about the excited
state properties of the sample. The anisotropy is affected by Brownian rotation, energy
transfer, re-absorption, re-emission and light scattering. Applications involve studying
asymmetric complex molecules, environmental perturbations, binding, hindered-
rotation phase transitions, and internal viscosities of bilayers.
Time-resolved acquisition
Time-resolved acquisition
scans measure the change in the spectral characteristics of
the sample during the lifetime of the excited state. The measurement consists of deter-
mining the frequency response of the sample over a specified emission range. Applica-
tions involve solvent relaxation of the excited state and excimer formation.
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