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AKB: Biologische Physik

AKB 40: Poster Session II

AKB 40.56: Poster

Mittwoch, 29. März 2006, 16:30–19:30, P3

Two-photon scanning fluorescence correlation and cross-correlation spectroscopy — •Zdeněk Petrášek and Petra Schwille — Biotechnologisches Zentrum der TU Dresden; Institut für Biophysik; Tatzberg 47 - 51; 01307 Dresden; Germany

Fluorescence correlation and cross-correlation spectroscopy methods (FCS and FCCS) obtain information about molecular diffusion and inter- and intramolecular processes by analysing fluctuations of the fluorescence intensity reflecting the fluctuations of various physical parameters. The fluctuations are quantified by means of the autocorrelation function of the measured signal F(t). In order for the autocorrelation to be representative of the investigated system the averaging in the autocorrelation calculation has to be performed over sufficiently high number of statistically independent events. This may be difficult to achieve when the diffusion of the fluorescent particles is slow, resulting in insufficient turnover of the particles in the measurement volume.

The scanning FCS (SFCS) combines the standard FCS with relative movement of the sample and the excitation beam. This improves the statistical accuracy by averaging over more independent locations within the sample. Furthermore, photobleaching effects can be reduced since the excitation dose is distributed over a larger part of the sample. We have employed a home-build two-photon laser scanning system to compare SFCS and SFCCS with their stationary counterparts, using several scanning patterns. The focus is on achieving a good signal-to-noise ratio and minimizing photobleaching effects while keeping the measurement time and the total light dose low.