Fluorescence

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 Timeresolved 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 timeresolved 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