Abstract
Förster Resonance Energy Transfer (FRET) allows for the visualization of nanometer-scale distances and distance changes. This sensitivity is regularly achieved in single-molecule experiments in vitro but is still challenging in biological materials. Despite many efforts, quantitative FRET in living samples is either restricted to specific instruments or limited by the complexity of the required analysis. With the recent development and expanding utilization of FRET-based biosensors, it becomes essential to allow biologists to produce quantitative results that can directly be compared. Here, we present a new calibration and analysis method allowing for quantitative FRET imaging in living cells with a simple fluorescence microscope. Aside from the spectral crosstalk corrections, two additional correction factors were defined from photophysical equations, describing the relative differences in excitation and detection efficiencies. The calibration is achieved in a single step, which renders the Quantitative Three-Image FRET (QuanTI-FRET) method extremely robust. The only requirement is a sample of known stoichiometry donor:acceptor, which is naturally the case for intramolecular FRET constructs. We show that QuanTI-FRET gives absolute FRET values, independent of the instrument or the expression level. Through the calculation of the stoichiometry, we assess the quality of the data thus making QuanTI-FRET usable confidently by non-specialists.
Highlights
Förster Resonance Energy Transfer (FRET) allows for the visualization of nanometer-scale distances and distance changes
Since the first cloning of the Green Fluorescent Protein (GFP), fluorescence microscopy has rapidly become a standard tool in cell biology
Förster Resonance Energy Transfer (FRET) methods circumvent this barrier by allowing the detection of distances below 10 nanometers between a donor fluorophore and an acceptor through non-radiative energy transfer mediated by dipole-dipole interactions
Summary
Förster Resonance Energy Transfer (FRET) allows for the visualization of nanometer-scale distances and distance changes. Fluorescence Lifetime Imaging Microscopy (FLIM) requires sophisticated instrumentation and analysis, and is often recognized as a quantitative method for live-cell measurements. It is generally accepted that bleedthrough of the donor emission in the acceptor channel and direct excitation of the acceptor by donor excitation channel must be corrected by substracting their contributions This requires the acquisition of three different signals, called 3-cube strategy in live-cell imaging[20]. To determine the correction factors and obtain as much information as possible from the sample, we follow a multiple excitation scheme as introduced by Kapanidis and colleagues for single molecule spectroscopy[18] and close to the three-cube method in live-cell imaging[19]. By switching rapidly between both excitation sources, and splitting the emission into two channels on the camera, we acquire in two successive snapshots four images: IDD: the detected signal in the donor channel after excitation at the donor wavelength, IDA: the detected signal in the acceptor channel after excitation at the donor wavelength, IAA: the detected signal in the acceptor channel after excitation at the acceptor wavelength
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
