12th Carl Zeiss sponsored workshop on ... - Institut Fresnel

Applications of scanning FCS
Zdeněk Petrášek
Biophysics group, Biotechnologisches Zentrum, Technische Universität Dresden, Tatzberg
47/49, 01307 Dresden, Germany
Scanning FCS (sFCS) is a modification of standard FCS where the probed volume is moved
across the sample in a defined way during the measurement. This is usually realized in a confocal
laser scanning microscope by scanning the beam in linear or circular fashion. Introduction of
scanning into the FCS measurements is motivated by different reasons, all of which aim to reduce
or remove particular limitations of standard FCS. These limitations include the problems
encountered with slowly moving molecules, such as poor statistical accuracy or photobleaching,
optical distortions of the measurement volume affecting the spatial calibration and the precision
of measurements, slow fluctuations of membranes disturbing the measurements on molecules
diffusing within the membrane, etc. Focusing on a different problem then leads to different sFCS
implementations. Here we present applications of sFCS employing circular scan paths with large
and small radii. While the former is applied when improvement of statistical accuracy and
reduction of photobleaching is desirable, the latter is particularly suitable in situations where
robust measurements of diffusion coefficients in the presence of optical distortions are the main
goal.