Fluorescence Correlation Spectroscopy (FCS) is a correlation analysis of temporal fluctuations of the fluorescence intensity. It offers insights into the photophysics that cause these characteristic fluorescence intensity fluctuations as well as diffusion behaviour and absolute concentrations of detected particles. The most prominent and easily observed cause of fluorescence fluctuations is the fluctuation of the concentration of fluorescent particles (molecules) in the obeservation volume. The method records temporal changes in the fluorescence emission intensity caused by single fluorophores passing the detection volume. These intensity changes can be quantified in their strength and duration by temporally auto-correlating the recorded intensity signal, leading to the average number of fluorescent particles in the detction volume and their average diffusion time through the volume. Eventually, important biochemical parameters as the concentration and size or shape of the particle (molecule) or viscosity of the environment can be determined.
FCS is a very sensitive analytical tool because it observes a small number of molecules (nanomolar to picomolar concentrations) in a small volume (fl). This concentration range fits to naturally occurring concentrations. Considering all the above, FCS is the perfect method to provide quantitative answers on diffusing molecules from within unperturbed compartments, like cells.
Fluorescence (Cross-) Correlation Spectroscopy (FCCS) is the daughter technique and correlates signal originating from two different fluorophores detected in two channels with each other. When two spectrally different fluorophores are attached to two molecules, dual-color-FCCS results in information of the degree of coinciding appearance in the optical volume. Through this we can learn about the degree of interaction between the fluorophores. FCCS therefore offers access to binding kinetics at low molecular concentrations in solution as well as unperturbed systems like living cells.
FCS can be performed with a continuous-wave laser, but using pulsed lasers allows even more sophisticated analysis possibilities like Fluorescence Lifetime Correlation Spectroscopy (FLCS) to elimate background or spectral crosstalk from the analysis. This is of paricaluar advantage when using spectrally inseperable fluorophores that differ in their life-time for FCCS. It also offers a way around afterpulsing artifacts.