Fluorescence Correlation and Cross-Correlation Spectroscopy Using Fluorescent Proteins for Measurements of Biomolecular Processes in Living Organisms

Fluorescence imaging and spectroscopy have played an important part in the advancement of the modern life sciences. Especially single molecule sensitive fluorescence techniques have allowed researchers to quantify biomolecular processes on the molecular level. However, these techniques were applied mainly in solution or in cell cultures that do not represent a physiologically relevant 3D environment. Results thus obtained are often not predictive of events in biomedically more interesting organisms or tissues. Therefore, many efforts are dedicated to bring single molecule fluorescence techniques into living model organisms. In this context, genetically encoded fluorescent proteins have played a major role. They exist in a wide variety (color, lifetime, Stokes shift, photoactivation, and environmental sensitivity) and ensure a 1:1 stoichiometry of labeling, important for the quantitation of data. In particular, two techniques, fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS), profited from these labels and were successfully modified for applications in organisms. In this chapter, we will review the basics of FCS and FCCS, the challenges of applying FCS in organisms, and the importance of fluorescent proteins in this process. We provide a discussion of the different applications to date and give a perspective of new developments with promising potential for applications within organisms. The application of FCS and other single molecule techniques within living organisms is an important step in the quantitation of biomolecular events in physiologically relevant 3D environments.