Research Papers: Imaging

Spectrally resolved fluorescence lifetime imaging microscopy: Förster resonant energy transfer global analysis with a one- and two-exponential donor model

[+] Author Affiliations
Daniela Strat, Frank Dolp, Angelika Rueck

University of Ulm, Institut for Lasertechnologies in Medicine, Helmholtzstrasse 12, Ulm, Baden-Wurttenberg 89081 Germany

Bjorn von Einem, Cornelia Steinmetz, Christine A. F. von Arnim

University of Ulm, Department of Experimental Neurology, 89081 Ulm, Germany

J. Biomed. Opt. 16(2), 026002 (February 10, 2011). doi:10.1117/1.3533318
History: Received July 21, 2010; Revised November 24, 2010; Accepted December 07, 2010; Published February 10, 2011; Online February 10, 2011
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In many fields of life science, visualization of spatial proximity, as an indicator of protein interactions in living cells, is of outstanding interest. A method to accomplish this is the measurement of Förster resonant energy transfer (FRET) by means of spectrally resolved fluorescence lifetime imaging microscopy. The fluorescence lifetime is calculated using a multiple-wavelength fitting routine. The donor profile is assumed first to have a monoexponential time-dependent behavior, and the acceptor decay profile is solved analytically. Later, the donor profile is assumed to have a two-exponential time-dependent behavior and the acceptor decay profile is derived analytically. We develop and apply a multispectral fluorescence lifetime imaging microscopy analysis system for FRET global analysis with time-resolved and spectrally resolved techniques, including information from donor and acceptor channels in contrast to using just a limited spectral data set from one detector only and a model accounting only for the donor signal. This analysis is used to demonstrate close vicinity of β-secretase (BACE) and GGA1, two proteins involved in Alzheimer's disease pathology. We attempt to verify if an improvement in calculating the donor lifetimes could be achieved when time-resolved and spectrally resolved techniques are simultaneously incorporated.

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© 2011 Society of Photo-Optical Instrumentation Engineers (SPIE)

Citation

Daniela Strat ; Frank Dolp ; Bjorn von Einem ; Cornelia Steinmetz ; Christine A. F. von Arnim, et al.
"Spectrally resolved fluorescence lifetime imaging microscopy: Förster resonant energy transfer global analysis with a one- and two-exponential donor model", J. Biomed. Opt. 16(2), 026002 (February 10, 2011). ; http://dx.doi.org/10.1117/1.3533318


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