Enhanced FRET contrast in lifetime imaging.
In combination with two photon excitation, FLIM is currently one of the best techniques to quantitatively study the subcellular localization of protein-protein interactions in living cells. An appropriate analysis procedure is crucial to obtain reliable results. TCSPC is an accurate method to measure FLIM. It is however an indirect process that requires photon decay curve fitting, using an exponential decay equation. Although choosing the number of exponential terms is essential, it is labor-intensive and time consuming. Therefore, a mono-model is usually applied to a whole image. Here we propose an algorithm, named Lichi, allowing pixel by pixel analysis based on the Deltachi(2) value. Lichi was validated using simulated photon decay curves with known lifetimes and proportions. It showed a high robustness for decay curves with more than 10(3) photons. When applied to lifetime images acquired from living cells, it resulted in a more realistic representation of the interaction maps. We developed an easy-to-use procedure for multi-model FLIM analysis, which enables optimized FRET quantification for all interaction texture studies, and is especially suitable to avoid the classical misinterpretation of heterogeneous samples.
Spriet
Corentin
C
Lille University of Science and Technology, Interdisciplinary Research Institute, CNRS USR 3078, Biophotonic team, 1 rue Prf. Calmette, 59021 Lille cedex, France.
Trinel
Dave
D
Riquet
Franck
F
Vandenbunder
Bernard
B
Usson
Yves
Y
Heliot
Laurent
L
D000465:Algorithms; D002470:Cell Survival / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008954:Models, Biological; D015739:Nocodazole / Q000494:pharmacology; D017785:Photons; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D020691:rab GTP-Binding Proteins / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D020691:rab GTP-Binding Proteins; D015739:Nocodazole