Imaging FRET standards by steady‐state fluorescence and lifetime methods

Abstract

Imaging fluorescence resonance energy transfer (FRET) between molecules labeled with fluorescent proteins is emerging as a powerful tool to study changes in ions, ligands, and molecular interactions in their physiological cellular environment. Different methods use either steady-state fluorescence properties or lifetime to quantify the FRET rate. In addition, some provide the absolute FRET efficiency whereas others are simply a relative index very much influenced by the actual settings and instrumentation used, which makes the interpretation of a given FRET rate very difficult. The use and exchange of FRET standards in laboratories using these techniques would help to overcome this drawback. We report here the construction and systematic evaluation of FRET standard probes of varying FRET efficiencies. The standards for intramolecular FRET were protein fusions of the cyan and yellow variants of A. victoria green fluorescent protein (ECFP and citrine) joined by short linkers or larger protein spacers, or ECFP tagged with a tetracysteine motif and labeled with the biarsenical fluorochrome, FlAsH. Negative and positive controls of intermolecular FRET were also used. We compared these FRET standards with up to four FRET quantification methods: ratioing of acceptor to donor emission, donor intensity recovery upon acceptor photobleach, sensitized emission after spectral unmixing of raw images, and fluorescence lifetime imaging (FLIM). The latter was obtained with a frequency-domain setup able to provide high quality lifetime images in less than a second, and is thus very well suited for live cell studies. The FRET rates or indexes of the standards were in good agreement regardless of the method used. For the CFP-tetraCys/FlAsH pair, the rate calculated from CFP quenching was faster than that obtained by FLIM.

High throughput FRET analysis of protein–protein interactions by slide‐based imaging laser scanning cytometry

“…In case only a fraction of donors is in complex with an acceptor, the gained FRET efficiency can be considered as a lower limit of the real E value. To resolve donor subpopulations characterized by different E values, fluorescence lifetime measurements (15)(16)(17) or single molecule conditions are needed (18,19).…”

Imaging local estrogen production in single living cells with recombinant fluorescent indicators

“…The FRET ratio (R) was a pixel by pixel division of a FRET channel image (obtained with donor excitation and acceptor emission) and a ECFP channel image (donor excitation and donor emission) and was displayed in pseudocolor (Domingo et al, 2007). FRET ratio change (R ) in time course experiments lasting 60-90 min and dose-response curves was obtained by dividing ratio at a given time (R) by ratio at time zero (R 0 , before addition of ligands), as:…”

Discrimination between alternate membrane protein topologies in living cells using GFP/YFP tagging and pH exchange

“…1.25 409 oil immersion objective, and a polychromator (C7773, Hamamatsu) for excitation (500 nm) were used [27]. FP construct-transfected cells were imaged with filtercube BrightLine HC-YFP (Semrock 500/24 nm exciter, 520 nm dichroic and a 542/ 27 nm emitter, center wavelength/bandwidth).…”