1. Fluorescence lifetime correlation spectroscopy A cross-talk free dual color fluorescence cross correlation spectroscopy Dual-Color Fluorescence Lifetime Correlation Spectroscopy to Quantify Protein–Protein Interactions in Live Cell. S. Padilla-Parra, N. Audugé, M. Coppey-Moisan & M. Tramier. Micros. Res. Tech. 74:788–793 (2011)

2. Main experimental artifact using GFP and mCherry in FCCS False-positive cross-correlation from cross-talk Wavelength (nm) 500600700 Fluorescence intensity Pulsed Interleaved Excitation Müller et al., Biophys J 2005 Thews et al., Biophys J 2005 We choose to use Fluorescence Lifetime Correlation Spectroscopy Green species contributing to red detection to overcome Switching method or Alternate Laser EXcitation Takahashi et al., Cell Struct Funct 2008 Miller et al., Anal Chem 2009 Combination of ALEX with Scanning FCS Ries et al., Nat Methods 2009

3. Microtime system from Picoquant for Dual Color FLCS 470nm pulsed laser 561nm continuous laser xyz piezo-scanning of the objective PC acquisition and analysis SPAD Perkin Elmer Picoharp 200 Confocal pinhole Live sample TTTR Mode

4. FLCS filter : how does it work? Fluorescence decay Lifetime filters Blue: decay Red: background Auto- and Cross-correlation using lifetime filters Böhmer et al., Chem Phys Lett 2002 Kapusta et al., J Fluoresc 2007

5. Dual color GFP and mCherry FLCS : methodology GFP excited with pulsed laser = decay filter on green detector Selecting photons coming from GFP fluorescence decay Removing background photons (detector after pulses, shot noise…) mCherry excited with continuous laser = background filter on red detector Selecting photons coming from mCherry Removing photons coming from GFP fluorescence decay Cross-correlation analysis using Symphotime software from Picoquant Cross-talk free cross-correlation curve

6. Dual color GFP and mCherry FLCS : tandem control GFP mCherry GFP mCherry Separate diffusion Co-diffusion 10 -2 10 -1 10 10 2 10 3 (ms) 10 -2 10 -1 10 2 10 3 (ms)