Fluorescence correlation techniques are used to investigate photophysical, photochemical, interaction and transport properties of fluorescent or fluorescently labelled molecules at extremely low concentrations by analyzing the fluctuations of the measured fluorescence signal. Since their introduction more than thirty years ago, many variations of fluorescence correlation techniques have been developed. They range from the original and the most widely applied Fluorescence Correlation Spectroscopy analyzing temporal fluctuations at a fixed position and suitable for the investigation of molecules in motion to Image Correlation Spectroscopy analyzing spatial correlations of immobile species. Scanning Fluorescence Correlation Spectroscopy is a group of correlation techniques where the measurement volume is moved across the sample in a defined way, resulting in a spatiotemporal correlation of the detected fluorescence. Scanning improves the accuracy of measurements on slowly moving molecules, diminishes the negative effects of photobleaching, and allows measurements on systems where other fluorescence correlation approaches perform poorly or are not possible. This chapter discusses scanning FCS in its relation to other fluorescence correlation methods, describes different variations of scanning FCS, summarizes some of the applications, and finally presents an example of experimental setup designed for two-photon scanning FCS.
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Petrášek, Z., Schwille, P. (2008). Scanning Fluorescence Correlation Spectroscopy. In: Rigler, R., Vogel, H. (eds) Single Molecules and Nanotechnology. Springer Series in Biophysics, vol 12. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-540-73924-1_4
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