Fluorescence Correlation Spectroscopy (FCS) / Fluorescence Lifetime Correlation Spectroscopy (FLCS)
Fluorescence Correlation Spectroscopy (FCS) measures the fluctuation in fluorescence over time due to fluorescent molecules moving in and out of an illuminated volume where the light is focused onto. From the time-trace of the fluorescent intensity one can subsequently perform an autocorrelation analysis. Fitting the autocorrelation curve will yield the Diffusion coefficient (which can also be used to estimate the size of molecules given some a priori information) and the concentration of molecules in the measured volume. The measurement is only performed on a single point either in vitro or in vivo.
Examples of use:
- Binding (dynamics) or colocalization of two fluorescently-labelled molecules separated (especially by more >10nm).
- Fluorescence Lifetime Imaging (FLIM) in Confocal Microscopy Applications: An Overview (Application Note by Picoquant GmbH)
- Fluorescence lifetime imaging microscopy: Spatial resolution of biochemical processes in the cell Bastiaens PIH, Squire A (1999)Trends in Cell Biology, 9:48-52.