Protein rotation in viscous environments can be measured by fluorescence depletion anisotropy (FDA) which combines long lifetimes of chromophore triplet states with the sensitivity of fluorescence excitation and detection. FDA achieves sensitivity well beyond that attainable by the more common technique of time-resolved phosphorescence anisotropy (TPA). We have now combined benefits of both time-domain and frequency-domain FDA into a single continuous technique (CFDA). Intensity and polarization of a single laser beam are modulated continuously according to a complex, repeating waveform. Fluorescence signals excited from triplet-forming fluorescent probes are digitized over recurring waveform periods by a high-speed signal averager. CFDA experiments typically involve substantial ground state depletion. Thus signals, unlike those of TPA, are not linear in the exciting light intensity and simple data analysis based on such linearity is not appropriate. An exact solution of the coupled diffusion and triplet production/decay equation describing CFDA within individual data points has been combined with simulated annealing optimization to extract triplet and anisotropy decay kinetics from experimental data. Related calculations compare possible excitation waveforms with respect to rotational information provided per fluorescence photon. We present CFDA results for the model system of eosin conjugates of carbonic anhydrase, BSA and immunoglobulin G in 90% glycerol at various temperatures and initial cellular results on eosin-IgE bound to 2H3 cell Type I Fcε receptors. We explore how CFDA reflects rotational parameters of heterogeneous systems and discuss challenges of extending this method to single cell microscopic measurements.
Keywords: Diffusion; Eosin; Phosphorescence; Triplet.