Fluorescence Correlation Spectroscopy: A Highly Sensitive Tool for Probing Intracellular Molecular Dynamics and Disease Diagnosis

Fluorescence correlation spectroscopy (FCS) is a very versatile and powerful technique that is based on time-averaging fluctuation analysis of fluorescence intensity generated in a tiny volume and can easily achieve single-molecule sensitivity. Briefly, in FCS, the fluctuations in fluorescence are recorded as a function of time and subsequently statistically analyzed by autocorrelation analysis. FCS helps to determine concentrations, diffusional dynamics, molecular interactions, intersystem crossing, and excited-state reactions of fluorescent species. Recent advances in related methods have pushed the frontiers such that FCS can now be applied to increasingly complex systems such as live cells and organisms to obtain quantitative data at physiological concentrations. In this chapter, we provide a brief overview of the basic principle of FCS, the experimental aspects, including the FCS data analysis, and emerging and efficient varieties of FCS that are currently being used to probe intracellular molecular dynamics and serve as a diagnostic tool for various disease conditions and characterization of biomedical samples. We intend to motivate the reader to appreciate the versatility of FCS as a tool being used in a plethora of disciplines ranging from photophysics to biophysical to biomedical sciences and across in vitro and in vivo systems.