Research Papers: Imaging

Application of phasor plot and autofluorescence correction for study of heterogeneous cell population

[+] Author Affiliations
Henryk Szmacinski

University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, Baltimore, Maryland 21201

Vladimir Toshchakov

University of Maryland School of Medicine, Department of Microbiology and Immunology, Baltimore, Maryland 21201

Joseph R. Lakowicz

University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, Baltimore, Maryland 21201

J. Biomed. Opt. 19(4), 046017 (Apr 25, 2014). doi:10.1117/1.JBO.19.4.046017
History: Received September 14, 2013; Revised March 9, 2014; Accepted March 25, 2014
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Abstract.  Protein-protein interactions in cells are often studied using fluorescence resonance energy transfer (FRET) phenomenon by fluorescence lifetime imaging microscopy (FLIM). Here, we demonstrate approaches to the quantitative analysis of FRET in cell population in a case complicated by a highly heterogeneous donor expression, multiexponential donor lifetime, large contribution of cell autofluorescence, and significant presence of unquenched donor molecules that do not interact with the acceptor due to low affinity of donor-acceptor binding. We applied a multifrequency phasor plot to visualize FRET FLIM data, developed a method for lifetime background correction, and performed a detailed time-resolved analysis using a biexponential model. These approaches were applied to study the interaction between the Toll Interleukin-1 receptor (TIR) domain of Toll-like receptor 4 (TLR4) and the decoy peptide 4BB. TLR4 was fused to Cerulean fluorescent protein (Cer) and 4BB peptide was labeled with Bodipy TMRX (BTX). Phasor displays for multifrequency FLIM data are presented. The analytical procedure for lifetime background correction is described and the effect of correction on FLIM data is demonstrated. The absolute FRET efficiency was determined based on the phasor plot display and multifrequency FLIM data analysis. The binding affinity between TLR4-Cer (donor) and decoy peptide 4BB-BTX (acceptor) was estimated in a heterogeneous HeLa cell population.

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© 2014 Society of Photo-Optical Instrumentation Engineers

Citation

Henryk Szmacinski ; Vladimir Toshchakov and Joseph R. Lakowicz
"Application of phasor plot and autofluorescence correction for study of heterogeneous cell population", J. Biomed. Opt. 19(4), 046017 (Apr 25, 2014). ; http://dx.doi.org/10.1117/1.JBO.19.4.046017


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