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Cross talk free fluorescence cross correlation spectroscopy in live cells
Elmar Thews1, Margarita Gerken, Reiner Eckert
1Institute of Physics and Department of Biophysics, Institute of Biology, University of Stuttgart, D-70550 Stuttgart, Germany.
Biophysical Journal
|June 14, 2005
Summary
This study introduces a novel fluorescence cross-correlation spectroscopy (FCCS) method using dual-color excitation to analyze protein binding in live cells. The technique successfully eliminates channel crosstalk, enabling accurate detection of molecular interactions.
Area of Science:
- Cell biology
- Biophysics
- Spectroscopy
Background:
- Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are powerful tools for studying biomolecules in live cells.
- Current FCCS applications using autofluorescent proteins (AFPs) are limited by detector channel crosstalk due to spectral overlap.
Purpose of the Study:
- To develop a new FCCS method for analyzing protein binding behavior in live cells.
- To overcome the limitations of spectral overlap in AFP-based FCCS.
Main Methods:
- Utilized dual-color excitation of enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) for discrimination.
- Employed pulsed excitation and detection on a timescale shorter than particle residence time to eliminate crosstalk.
- Validated the method using ECFP/EYFP lysates, chimeras, and live HeLa cells coexpressing ECFP-connexin and EYFP-connexin fusion proteins.
Main Results:
- Successfully eliminated crosstalk between detector channels.
- Obtained an undisturbed cross-correlation signal.
- Demonstrated the method's efficacy in analyzing protein interactions in live cells.
Conclusions:
- The novel dual-color excitation FCCS method effectively analyzes protein binding in live cells.
- This technique overcomes previous crosstalk limitations, providing a more accurate tool for cell biology research.