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BP: Fachverband Biologische Physik

BP 2: New Technologies

BP 2.3: Vortrag

Montag, 22. März 2010, 11:00–11:15, H43

Robust measurement of diffusion with scanning FCS — •Zdeněk Petrášek, Susan Derenko, and Petra Schwille — Biophysics group, Biotechnologisches Zentrum, Technische Universität Dresden, Tatzberg 47/49, 01307 Dresden, Germany

We present a simple modification of Fluorescence Correlation Spectroscopy (FCS), where the position of the measurement volume is not stationary, but is scanned along a circle of a diameter comparable to the volume size. The known scan radius serves as an internal spatial standard, meaning that the exact volume size need not be known to determine the diffusion coefficient. In this way the method gains robustness against optical distortions that may uncontrollably enlarge the measurement volume, or affect its shape.

The oscillations, introduced into the measured correlation curve by scanning, favourably constrain the fit of a model to the data. Therefore, a relatively narrow range of the autocorrelation is sufficient to obtain a stable fit. Consequently, the correlation values at short lag times, that may be influenced by complicated photophysics of the dye or suffer from a poor signal-to-noise ratio, or the values at long correlation times, that may be affected, for example, by depletion due to photobleaching, can be excluded from the analysis, contributing to the robustness of the method.

The implementation of scanning FCS based on a two-dimensional piezo scanner has been applied to studies both in bulk solution and on surfaces — supported lipid bilayers and giant unilamellar vesicles.