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Fluorescence Correlation Spectroscopy (FCS): Theory and Applications

The practical course will focus on protein diffusion measurements performed on living bacterial cells. We will learn how to extract information from the fluctuations of fluorescence intensity signals acquired from biological samples.

The following concepts and approaches will be explained:

1) Jablonski diagram of a fluorescent dye.
2) Optical resolution and optical aberrations.
3) Functioning of a laser scanning confocal microscope.
4) Calculation of the autocorrelation functions of a fluorescence intensity signal.
5) Diffusion models for analyzing fluorescence correlation spectroscopy data acquired in living cells
6) Dedicated software packages for data analysis of FCS measurements.

The course will involve a practical part where measurements will be acquired using a Zeiss LSM880 confocal microscope and a data analysis part in which we will use the Zeiss proprietary software ZEN and the freely available software ImageJ (NIH) and QuickFit (J.Langowski, DKFZ, Heidelberg).