Regensburg 2007 – scientific programme

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BP: Fachverband Biologische Physik

BP 16: Poster Session I

BP 16.41: Poster

Tuesday, March 27, 2007, 17:00–19:30, Poster D

Photobleaching in two-photon scanning fluorescence correlation spectroscopy — •Zdeněk Petrášek and Petra Schwille — Biotechnologisches Zentrum, Institut für Biophysik, TU Dresden

Two-photon Fluorescence Correlation Spectroscopy (FCS) takes advantage of the excitation being sufficiently localized, so that no confocal arrangement using a pinhole is necessary to create a well confined measurement volume. Although two-photon FCS has all the advantages well known from two-photon microscopy, signal-to-noise ratios lower than with one-photon excitation are usually achieved, a fact commonly attributed to optical saturation and photobleaching.

Scanning FCS (sFCS) combines the standard FCS with relative movement of the sample and the excitation beam. Although the information about diffusion kinetics is partially lost by scanning, sFCS can provide useful information on the role of photobleaching and saturation at high excitation intensities.

The measurements with circular-scanning sFCS indicate that photobleaching is the major factor responsible for the effects encountered at high excitation intensities, such as apparently shorter diffusion times and decrease of the autocorrelation amplitude g(0). Furthermore, sFCS reduces these effects and extends the range of excitation intensities where g(0) is not affected by photobleaching, thus allowing measurements at higher molecular brightness and S/N ratio.

Numerical calculations show that photobleaching alone can explain the observed decrease of g(0) and lower than expected fluorescence, without the need to consider saturation effects.