Berlin 2005 – wissenschaftliches Programm

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AKB: Biologische Physik

AKB 100: Poster Session I

AKB 100.58: Poster

Samstag, 5. März 2005, 16:45–18:45, Poster TU D

Cross-talk Free Fluorescence Cross Correlation Spectroscopy in Living Cells — •Andrew Aird¹, Elmar Thews¹, Carsten Tietz¹, Reiner Eckert², and Jörg Wrachtrup¹ — ¹Institute of Physics, University of Stuttgart, Pfaffenwaldring 57, D?70550 Stuttgart — ²Department of Biophysics, Institute of Biology, University of Stuttgart, Pfaffenwaldring 57, D?70550 Stuttgart

Fluorescence correlation spectroscopy (FCS) is now a widely used technique to measure small ensembles of labeled biomolecules even in living cells. Fluorescence cross correlation spectroscopy (FCCS) is more suited to detect synchronous movement of two biomolecules with different labels and so grants access to a wide variability of unsolved questions in cell biology. Autofluorescent proteins are labels being less cell perturbing in its appliance and highly specific in binding to the proteins in question. The method presented here fuses the advantages of these three techniques to analyze binding behavior of proteins in living cells. To achieve this, a common pair of autofluorescent proteins CFP and YFP is discriminated rather in excitation than in fluorescence to eliminate cross-talk in the detector channels and obtain an undisturbed cross correlation function. The setup is tested to work in living HeLa cells coexpressing the two fusion proteins Cx46/CFP and Cx46/YFP, which form hetero-labeled hexamers (connexones) and diffuse freely in the plasma membrane.