期刊
MICROSCOPY RESEARCH AND TECHNIQUE
卷 74, 期 8, 页码 788-793出版社
WILEY
DOI: 10.1002/jemt.21015
关键词
FCS; FCCS; FLCS; quantitative fluorescence microscopy; TCSPC
资金
- Fondation pour la Recherche Medicale
- Region Ile de France (Soutien aux Equipes Scientifiques pour l'Acquisition de Moyens Experimentaux)
- Groupement Entreprises Francaises Lutte Contre Cancer
- Centre National de la Recherche Scientifique (Action Concertee Incitative Biologie Cellulaire, Moleculaire et Structurale)
- Association pour la Recherche sur le Cancer
- Association Nationale pour le Recherche
Dual-color fluorescence correlation spectroscopy is an interesting method to quantify protein interaction in living cells. But, when performing these experiments, one must compensate for a known spectral bleed through artifact that corrupts cross-correlation data. In this article, problems with crosstalk were overcome with an approach based on fluorescence lifetime correlation spectroscopy (FLCS). We show that FLCS applied to dual-color EGFP and mCherry cross-correlation allows the determination of protein-protein interactions in living cells without the need of spectral bleed through calibration. The methodology was validated by using EGFP-mCherry tandem in comparison with coexpressed EGFP and mCherry in live cell. The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during experiment is really very helpful to study quantitatively protein interactions in live sample. Microsc. Res. Tech. 74:788-793, 2011. (C) 2011 Wiley-Liss, Inc.
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