Spectrally Resolved Frequency Domain Analysis of Multi-Fluorophore Systems Undergoing Energy Transfer

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Abstract:

Complex systems of fluorophores undergoing energy transfer can exhibit a variety of anomalous lifetime behavior when probed with frequency domain methods. When presented in traditional apparent lifetime format the data from such systems can exhibit "nodal" behavior in which the computed lifetime approaches ±∞. The location of the nodes is system and frequency dependent. In addition, simpler systems, not undergoing energy transfer, show ill behavior in the region of zero lifetime (τ m ) and long lifetime (τπ) due to noise in typical measurements. Here, we systematically investigate systems of multiple fluorophores with and without energy transfer to provide insight into frequency domain investigations of complex systems of fluorophores. The results of simulations are compared to data collected from a multi-fluorophore system designed to exhibit fluorescence resonance energy transfer (FRET) using imaging spectroscopic fluorescence lifetime imaging microscopy (ISFLIM). The results are applicable to both cuvette and imaging arrangements.

Keywords: ENERGY TRANSFER; FLIM; FLUORESCENCE LIFETIME IMAGING MICROSCOPY; FLUORESCENCE LIFETIMES; FLUORESCENCE RESONANCE ENERGY TRANSFER; FRET; MULTI-FLUOROPHORE SYSTEMS; SFLIM; SPECTROSCOPIC FLUORESCENCE LIFETIME IMAGING MICROSCOPY

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/000370206779321544

Affiliations: 1: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1; Department of Biological and Chemical Sciences, University of the West Indies, Cave Hill Campus, St. Michael, Barbados 2: School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, United Kingdom; Department of Biological and Chemical Sciences, University of the West Indies, Cave Hill Campus, St. Michael, Barbados

Publication date: December 1, 2006

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