Correcting for spectral cross-talk in dual-color fluorescence cross-correlation spectroscopy.

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry
Kirsten BaciaP Schwille

Abstract

Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) allows one to quantitatively assess the interactions of mobile molecules labeled with distinct fluorophores. The technique is widely applied to both reconstituted and live-cell biological systems. A major drawback of dcFCCS is the risk of an artifactual false-positive or overestimated cross-correlation amplitude arising from spectral cross-talk. Cross-talk can be reduced or prevented by fast alternating excitation, but the technology is not easily implemented in standard commercial setups. An experimental strategy is devised that does not require specialized hardware and software for recognizing and correcting for cross-talk in standard dcFCCS. The dependence of the cross-talk on particle concentrations and brightnesses is quantitatively confirmed. Moreover, it is straightforward to quantitatively correct for cross-talk using quickly accessible parameters, that is, the measured (apparent) fluorescence count rates and correlation amplitudes. Only the bleed-through ratio needs to be determined in a calibration measurement. Finally, the limitations of cross-talk correction and its influence on experimental error are explored.

References

Mar 21, 1998·Proceedings of the National Academy of Sciences of the United States of America·U KettlingM Eigen
Mar 1, 1989·Physical Review A: General Physics·J Rika, T Binkert
Sep 6, 2000·Proceedings of the National Academy of Sciences of the United States of America·K G HeinzeP Schwille
Sep 5, 2002·Proceedings of the National Academy of Sciences of the United States of America·Tobias KohlPetra Schwille
Dec 30, 2003·Proceedings of the National Academy of Sciences of the United States of America·Sally A KimPetra Schwille
Oct 12, 2004·Biochemical and Biophysical Research Communications·Kenta SaitoMasataka Kinjo
Jun 14, 2005·Biophysical Journal·Elmar ThewsJörg Wrachtrup
Aug 23, 2005·Biophysical Journal·Barbara K MüllerDon C Lamb
Jan 25, 2006·Nature Methods·Kirsten BaciaPetra Schwille
Nov 17, 2007·Nature Protocols·Kirsten Bacia, Petra Schwille
Dec 14, 2007·Proceedings of the National Academy of Sciences of the United States of America·Brian D SlaughterRong Li
Jul 20, 2011·Current Opinion in Structural Biology·James A J Fitzpatrick, Björn F Lillemeier
Aug 13, 2011·Methods in Molecular Biology·Mark A HinkAntonie J W G Visser
Sep 6, 2011·Biophysical Journal·Johan StrömqvistJerker Widengren

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Citations

Dec 4, 2014·Biophysical Journal·Hetvi GandhiThomas Weidemann
Mar 10, 2016·The Journal of Cell Biology·Mayumi IsokaneJan Ellenberg
Nov 6, 2015·Nature Protocols·Jan W KriegerThorsten Wohland
Aug 16, 2014·Current Opinion in Structural Biology·Thomas WeidemannPetra Schwille
Aug 6, 2016·Journal of the American Chemical Society·Rabindra V ShivnaraineClaudiu C Gradinaru
Jun 15, 2016·PloS One·Daria Kotlarek, Remigiusz Worch
Nov 30, 2018·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Stefan WernerKirsten Bacia
Aug 10, 2017·Nature Communications·Julian PetersenGunnar Schulte
Mar 4, 2020·PLoS Computational Biology·Sebastian M BernasekLuís A N Amaral
Dec 5, 2018·Nature Communications·Syed Mukhtar AhmedIan G Macara
Jun 11, 2020·Methods and Applications in Fluorescence·Michael R StonemanValerică Raicu
Dec 21, 2018·The Journal of Physical Chemistry. B·Ioannis SgouralisSteve Pressé

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Methods Mentioned

BETA
FCS
fluorescence resonance
confocal microscopy
FRET

Software Mentioned

ConfoCor3
ConfoCor2

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