INTRODUCTION TO

FLUORESCENCE
 Fluorescence anisotropy
fluorescence anisotropy assays the rotational
diffusion of a molecule from the decorrelation of
polarization between the exciting and emitted
(fluorescent) photons.
Anisotropy

polarization

Several phenomena ca
decrease the measured
anisotropy to values lower
than the maximum
theoretical values. The
most common cause is
rotational diffusion
 Expected anisotropy

τfl >> θrot 

depolarized
τfl << θrot  polarized

Size and shape of proteins or the rigidity of various molecular environments.

Resonance Energy Transfer

This process occurs whenever

the emission spectrum of a
fluorophore, called the donor,
overlaps with the absorption
spectrum of another molecule,
called the acceptor.
The rate of energy transfer
kT(r)

The efficiency of energy transfer

for a single donor-acceptor pair at
a fixed distance
 Steady-state and Time-
resolved fluorescence
-Steady-state measurement
The sample is illuminated with a continuous beam of light, and the
intensity of emission spectrum is recorded
-Time-resolved measurement
The sample is exposed to a pulse of light, where the pulse width is
typically shorter than the decay time of the sample

A steady-state observation is simply an average of the time-

resolved phenomena over the intensity decay of the sample.

Time-resolved intensity and anisotropy Steady-state anisotropy

Why Time-Resolved
Measurements?
 Much of the molecular information
available from fluorescence is lost during
the time averaging process.
2) Shape information is lost during

averaging of the anisotropy over decay

time
3) The intensity decays also contain

information that is during the averaging

process
4) Much of the molecular information
content is available only by time-