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)

Main experimental artifact using GFP and mCherry in FCCS

False-positive cross-correlation from cross-talk

Wavelength (nm)

500 600 700

Fluo

resc

ence

inte

nsity

Pulsed Interleaved Excitation Müller et al., Biophys J 2005Thews et al., Biophys J 2005

We choose to use Fluorescence Lifetime Correlation Spectroscopy

Green species contributing to red detectionto overcome

Switching method or Alternate Laser EXcitationTakahashi et al., Cell Struct Funct 2008

Miller et al., Anal Chem 2009

Combination of ALEX with Scanning FCSRies et al., Nat Methods 2009

Microtime system from Picoquant for Dual Color FLCS

470nm pulsed laser

561nm continuous laser

xyz piezo-scanning of the objective

PC acquisitionand analysis

SPAD Perkin Elmer

SPAD Perkin ElmerPicoharp 200

Confocal pinhole

Live sample

TTTR Mode

FLCS filter : how does it work?

Fluorescence decay Lifetime filters

Blue: decay

Red: background

Auto- and Cross-correlation usinglifetime filters

Böhmer et al., Chem Phys Lett 2002Kapusta et al., J Fluoresc 2007

Dual color GFP and mCherry FLCS : methodology

GFP excited with pulsed laser = decay filter on green detectorSelecting photons coming from GFP fluorescence decay

Removing background photons (detector after pulses, shot noise…)

mCherry excited with continuous laser = background filter on red detectorSelecting photons coming from mCherry

Removing photons coming from GFP fluorescence decay

Cross-correlation analysisusing Symphotime software

from Picoquant

Cross-talk free cross-correlation curve

30x10-3

20

10

0

G(t

au)

log time

Auto- and Cross Correlation of GFP and mCherry co-express in live cell

GFP autocorrelation mCherry autocorrelation "classic" Crosscorrelation Lifetime filter Crosscorrelation

20x10-3

15

10

5

0

G(t

au)

log time

Auto- and Cross Correlation of GFP-mCherry tandem co-express in live cell

GFP autocorrelation mCherry autocorrelation "classic" Crosscorrelation Lifetime filter Crosscorrelation

Dual color GFP and mCherry FLCS : tandem control

GFP

mCherry

GFP

mCherry

Separate diffusion

Co-diffusion

10-2 10-1 10 102 103(ms)

10-2 10-1 102 103(ms)