Building and Using a Two-Photon Fluorescence Cross-Correlation Spectroscopy Setup Including Fluorescence Lifetime Analysis.

Fluorescence Cross-Correlation Spectroscopy (FCCS) is a well-established and useful tool in physics and chemistry. Furthermore, due to its hybrid character of being a bulk assay at a single molecular level, it found many applications in biophysics and molecular biochemistry. Examples may be investigating kinetics and dynamics of chemical and biochemical reactions such as protein-ligand-, protein-protein-binding, fast conformational changes, and intracellular transportation. Also, it was utilized to characterize larger structures such as lipid vesicles and multi-protein complexes. A two-photon excitation source makes FCCS relatively easy-to-use and easy-to-maintain. Combining this technique with fluorescence lifetime analysis results in a versatile biophysical tool that can be used to solve many biological problems, as even small changes in the local environment, like pH or salt concentration, can be monitored if appropriate fluorophores are used. An example of its use for membrane docking and fusion assays is described in Chap. 13 . In this chapter, we want to give the reader a simple, detailed step-by-step guide of how to set up such a tool.