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Fluorescence lifetime correlation spectroscopy

Fluorescence lifetime correlation spectroscopy. A cross-talk free dual color fluorescence cross correlation spectroscopy.

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Fluorescence lifetime correlation spectroscopy

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  1. Fluorescence lifetimecorrelationspectroscopy A cross-talk free dual color fluorescence cross correlationspectroscopy Dual-Color Fluorescence LifetimeCorrelationSpectroscopy to QuantifyProtein–Protein Interactions in Live Cell. S. Padilla-Parra, N. Audugé, M. Coppey-Moisan & M. Tramier. Micros. Res. Tech. 74:788–793 (2011)

  2. 500 600 700 Main experimental artifact using GFP and mCherry in FCCS Green speciescontributing to reddetection to overcome Pulsed Interleaved Excitation Müller et al., Biophys J 2005 Thews et al., Biophys J 2005 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 Fluorescence intensity False-positive cross-correlationfrom cross-talk Wechoose to use Fluorescence LifetimeCorrelationSpectroscopy Wavelength (nm)

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

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

  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 mCherry GFP Separate diffusion 10-1 10-2 (ms) 103 102 10 mCherry GFP Co-diffusion (ms) 10-1 10-2 103 102

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