title: Spectrally resolved fluorescence lifetime imaging microscopy: Förster resonant energy transfer global analysis with a one- and two-exponential donor model.
abstract: 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.
authors.affiliation: University of Ulm, Institut for Lasertechnologies in Medicine, Helmholtzstrasse 12, Ulm, Baden-Wurttenberg 89081, Germany. danastrat@yahoo.com
authors.forename: Daniela
authors.initials: D
authors.lastname: Strat
authors.forename: Frank
authors.initials: F
authors.lastname: Dolp
authors.forename: Bjorn
authors.initials: B
authors.lastname: von Einem
authors.forename: Cornelia
authors.initials: C
authors.lastname: Steinmetz
authors.forename: Christine A F
authors.initials: CA
authors.lastname: von Arnim
authors.forename: Angelika
authors.initials: A
authors.lastname: Rueck
mesh_terms: D004867:Equipment Design; D019544:Equipment Failure Analysis; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D017785:Photons; D025941:Protein Interaction Mapping / Q000295:instrumentation*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*