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
Green species contributing to red detection
Fluorescence intensity
to overcome
Pulsed Interleaved Excitation
Müller et al., Biophys J 2005
Thews et al., Biophys J 2005
Switching method or Alternate Laser EXcitation
500
600
700
Takahashi et al., Cell Struct Funct 2008
Miller et al., Anal Chem 2009
Wavelength (nm)
Combination of ALEX with Scanning FCS
Ries et al., Nat Methods 2009
False-positive cross-correlation from cross-talk
We choose to use Fluorescence Lifetime Correlation Spectroscopy
Microtime system from Picoquant for Dual Color FLCS
Live sample
TTTR Mode
xyz piezo-scanning of the objective
470nm pulsed laser
561nm continuous laser
Confocal pinhole
SPAD Perkin Elmer
PC acquisition
and analysis
Picoharp 200
SPAD Perkin Elmer
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
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
Dual color GFP and mCherry FLCS : tandem control
30x10
Auto- and Cross Correlation
of GFP and mCherry co-express in live cell
GFP autocorrelation
mCherry autocorrelation
"classic" Crosscorrelation
Lifetime filter Crosscorrelation
-3
G(tau)
20
mCherry
GFP
10
Separate diffusion
0
10-2
20x10
10-1
102
103
-3
Auto- and Cross Correlation
of GFP-mCherry tandem co-express in live cell
GFP autocorrelation
mCherry autocorrelation
"classic" Crosscorrelation
Lifetime filter Crosscorrelation
15
G(tau)
log time (ms) 10
10
mCherry
GFP
5
0
Co-diffusion
10-2
10-1
log time(ms)
102
103