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Setup and characterization of a multiphoton FLIM instrument for protein–protein interaction measurements in living cells
Author(s) -
Waharte François,
Spriet Corentin,
Héliot Laurent
Publication year - 2006
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20240
Subject(s) - förster resonance energy transfer , characterization (materials science) , fluorophore , fluorescence lifetime imaging microscopy , fluorescence , confocal , confocal microscopy , protocol (science) , biological system , computer science , materials science , nanotechnology , optics , physics , biology , medicine , alternative medicine , pathology
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. © 2006 Wiley‐Liss, Inc.