Cross talk free fluorescence cross correlation spectroscopy in live cells.
Fluorescence correlation spectroscopy (FCS) is now a widely used technique to measure small ensembles of labeled biomolecules with single molecule detection sensitivity (e.g., low endogenous concentrations). Fluorescence cross correlation spectroscopy (FCCS) is a derivative of this technique that detects the synchronous movement of two biomolecules with different fluorescence labels. Both methods can be applied to live cells and, therefore, can be used to address a variety of unsolved questions in cell biology. Applications of FCCS with autofluorescent proteins (AFPs) have been hampered so far by cross talk between the detector channels due to the large spectral overlap of the fluorophores. Here we present a new method that combines advantages of these techniques to analyze binding behavior of proteins in live cells. To achieve this, we have used dual color excitation of a common pair of AFPs, ECFP and EYFP, being discriminated in excitation rather than in emission. This is made possible by pulsed excitation and detection on a shorter timescale compared to the average residence time of particles in the FCS volume element. By this technique we were able to eliminate cross talk in the detector channels and obtain an undisturbed cross correlation signal. The setup was tested with ECFP/EYFP lysates as well as chimeras as negative and positive controls and demonstrated to work in live HeLa cells coexpressing the two fusion proteins ECFP-connexin and EYFP-connexin.
Thews
Elmar
E
Institute of Physics and Department of Biophysics, Institute of Biology, University of Stuttgart, D-70550 Stuttgart, Germany.
Gerken
Margarita
M
Eckert
Reiner
R
Zäpfel
Johannes
J
Tietz
Carsten
C
Wrachtrup
Jörg
J
D001426:Bacterial Proteins / Q000737:chemistry; D003470:Culture Media / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D015233:Models, Statistical; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014162:Transfection
D001426:Bacterial Proteins; D003470:Culture Media; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins