**Fluorescence correlation spectroscopy**

Fluorescence correlation spectroscopy (FCS) records fluctuations of fluorescence intensity over time in a confocal observation volume using highly-sensitive avalanche diodes for detection. These fluctuations are caused by fluorescently labeled molecules diffusing through the observation volume, photophysics of the fluorophores, or chemical reactions. An autocorrelation function can be determined (figure 1a) and fitted yielding information on these processes, such as diffusion coefficients, particle concentrations, rate constants of various types of kinetics, or dissociation constants (figure 1b). While conventional FCS is a useful tool to study samples in solution with a high temporal resolution it does not give any information on the spatial heterogeneity of a biological sample.

**Figure 1: Principle and applications of FCS.** (a) Recording of fluctuations of fluorescence intensity in a confocal volume facilitates the calculation of an autocorrelation function which yields information on the observed biological target system. (b) Determination of dissociation constant of the receptor tyrosine kinase MET and its ligand InlB by analysis of FCS data.

Imaging FCS (imFCS) is a camera-based method adapting the principles of FCS to multiple dimensions. It applies TIRF illumination facilitating imaging of an extended region of interest of a biological sample. Intensity fluctuations and autocorrelation functions are determined for each pixel of the observed area (figure 1a). Thus, a single measurement yields hundreds of data points enabling statistical analysis (figure 1b). Maps of concentrations and diffusion coefficients can be created depicting the heterogeneity of a sample (figure 1c).

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**Figure 2: Principle and applications of imFCS.** (a) A region of interest in a cell is selected for imFCS measurements. For each pixel of this region an autocorrelation function is calculated and fitted. (b) By analyzing the autocorrelation functions an average distribution of diffusion coefficients can be determined for the sample. (c) Maps of concentration and diffusion coefficients can be obtained yielding spatial information on the heterogeneity.