The polarized AB plot for the frequency-domain analysis and representation of fluorophore rotation and resonance energy homotransfer

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Title: The polarized AB plot for the frequency-domain analysis and representation of fluorophore rotation and resonance energy homotransfer
Author(s): Clayton, A. H. A.
Abstract: The graphical representation of single-frequency phase-modulation fluorescence lifetime imaging data, referred to as the AB plot, is extended to take into account measurements of the polarized components of the fluorescence. For a hindered rotator model (characterized with a single excited-state lifetime, a single rotational correlation time and limiting initial and final anisotropies) the rotational correlation time and the excited lifetime can be determined from the AB plot of any two of the following emission components: parallel, perpendicular, total emission or combinations thereof. A strategy for resolving the component hindered rotations and lifetimes for mixtures of two hindered rotators from measurements of the total, parallel and perpendicular components of the emission is developed. The analysis does not require prior knowledge of the initial limiting anisotropy or of the steady-state anisotropy or of the excited state lifetime. Plots in polarized AB space derived for heterogeneous systems are constructed to aid interpretation of frequency-domain dynamic depolarization imaging microscopy experiments. These plots can be used to distinguish spatially dependent rotational correlation time heterogeneity from heterogeneity in limiting anisotropies. The effects of noise and aperture depolarization are discussed. It is anticipated that the polarized AB plot will provide a useful adjunct to existing methods for visualizing and analysing dynamic polarization phenomena arising from molecular dynamics and homo-energy transfer in single-frequency microscopy applications.
Publication type: Journal article
Source: Journal of Microscopy, Vol. 232, no. 2 (Nov 2008), pp. 306-312
Publication year: 2008
FOR Code(s): 0204 Condensed Matter Physics; 0912 Materials Engineering
Keyword(s): Anisotropy; Correlation time; Energy transfer; Field emission; FLIM; Fluorescence microscopy; Fluorescence polarisation; Polarization; rFLIM
Publisher: Wiley-Blackwell Publishing
ISSN: 0022-2720
Publisher URL: http://dx.doi.org/10.1111/j.1365-2818.2008.02102.x
Peer reviewed