Setup and characterization of a multiphoton FLIM instrument for protein-protein interaction measurements in living cells.
BACKGROUND
Fluorescence lifetime microscopy (FLIM) is currently one of the best techniques to perform accurate measurements of interactions in living cells. It is independent of the fluorophore concentration, thus avoiding several common artifacts found in Förster Resonance Energy Transfer (FRET) imaging. However, for FLIM to achieve high performance, a rigorous instrumental setup and characterization is needed.


METHODS
We use known fluorophores to perform characterization experiments in our instrumental setup. This allows us to verify the accuracy of the fluorescence lifetime determination, and test the linearity of the instrument by fluorescence quenching.


RESULTS
We develop and validate here a protocol for rigorous characterization of time-domain FLIM instruments. Following this protocol, we show that our system provides accurate and reproducible measurements. We also used HeLa cells expressing proteins fused to Green Fluorescent Proteins variants (CFP and YFP) to confirm its ability to detect interactions in living cells by FRET.


CONCLUSIONS
We report a well-designed protocol in which precise and reproducible lifetime measurements can be performed. It is usable for all confocal-based FLIM instruments and is a useful tool for anyone who wants to perform quantitative lifetime measurements, especially when studying interactions in living cells using FRET.
Waharte
François
F
Laboratoire d'Imagerie Cellulaire Fonctionnelle, Institut de Biologie de Lille/Institut Pasteur Lille, Lille, France.
Spriet
Corentin
C
Héliot
Laurent
L
D001426:Bacterial Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods*; D011485:Protein Binding; D015203:Reproducibility of Results
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins