Dresden 2009 – scientific programme

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BP: Fachverband Biologische Physik

BP 7: Poster I

BP 7.2: Poster

Monday, March 23, 2009, 17:45–20:00, P3

Scanning Fluorescence Correlation Spectroscopy on Membranes — •Jonas Ries, Salvatore Chiantia, Rachel Yu, and Petra Schwille — Biotec, TU Dresden, Tatzberg 47-51, 01307 Dresden, Germany

When confocal fluorescence correlation spectroscopy (FCS) is applied on membranes, long measurement times are required and instabilities, photobleaching or poor knowledge of the detection area limit the accuracy. Here we present two implementations of scanning FCS (SFCS) to circumvent these problems. Scanning FCS with a scan path perpendicular to the membrane plane is robust against instabilities and allows for very long measurement times, which are required to study slow diffusion. It can be extended to measure calibration-free diffusion constants with scanning two focus FCS and to quantify binding on the membrane with scanning dual color FCS with alternating excitation. We applied this method to study the affinity of the Fgfr1(4) to its ligand Fgf8 in the membranes of living zebra fish embryos. Line-scan FCS with a scan path parallel to the membrane plane greatly increases the statistics by parallel acquisition. It allows for calibration-free diffusion and concentration measurements on membranes within seconds and is virtually not affected by photobleaching. Both approaches can be easily implemented with commercial laser scanning microscopes and allow for quantitative measurements in demanding systems previously not accessible by FCS.