Quantitative Study of protein-protein interactions in live cell by dual-color fluorescence correlation spectroscopy

Sergi Padilla-Parra, Nicolas Audugé, Maïté Coppey-Moisan, Marc Tramier

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

7 Citations (Scopus)


Dual-color FCS is a powerful method to monitor protein-protein interactions in living cells. The main idea is based on the cross-correlation analysis of temporal fluorescence intensity fluctuations of two fluorescent proteins to obtain their co-diffusion and relative concentration. But, when performing these experiments, the spectral overlap in the emission of the two colors produces an artifact that corrupts the cross-correlation data: spectral bleed-through. We have shown that problems with cross talk are overcome with Fluorescence Lifetime Correlation Spectroscopy (FLCS). FLCS applied to dual-color cross-correlation, utilizing for example eGFP and mCherry fluorescent proteins, allows the determination of protein-protein interactions in living cells without the need of spectral bleed-through calibration. Here, we present in detail how this methodology can be implemented using a commercial setup (Microtime from PicoQuant, SP8 SMD from Leica or any conventional confocal with PicoQuant TCSPC module, and also with a Becker and Hickl TCSPC module). The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during the experiment constitutes a very powerful technique to quantitatively study protein interactions in live samples.

Original languageEnglish
Title of host publicationFluorescence Spectroscopy and Microscopy
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc
Number of pages16
ISBN (Print)9781627036481
Publication statusPublished - 2014

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745


  • FCCS
  • FCS
  • FLCS
  • Quantitative fluorescence microscopy


Dive into the research topics of 'Quantitative Study of protein-protein interactions in live cell by dual-color fluorescence correlation spectroscopy'. Together they form a unique fingerprint.

Cite this