Fluorescence correlation spectroscopy (FSC) and raster image correlation spectroscopy (RICS)

About this technique

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Fluorescence correlation spectroscopy (FCS)

FCS is a highly sensitive technique that measures fluctuations in the intensity of fluorescence over time. It can be used to study the interactions and relative movements of fluorescent or fluorescently-labeled molecules due to diffusion, or physical or chemical interactions. It provides extremely detailed time-resolved data from a given area of interest in the nanosecond- to millisecond-range with single-molecule sensitivity. It therefore allows the behaviour of molecules to be observed in living cells. It is usually used on a confocal or multiphoton microscope.

Because fluctuations in the signal are measured, the technique works best when the numbers of labeled molecules in the sample area are small. In this way, changes in the number of molecules in the target area are significant as a proportion of the total fluorescence present. This problem needs to be balanced with the opposite situation, where too few molecules enter or exit from the target area to give any significant fluctuations and therefore, meaningful results. By using differently coloured fluorescent markers, several species of molecule can be observed simultaneously.

When optimised, FCS can be used to obtain quantitative information on properties such as:

• diffusion coefficients
• hydrodynamic radii
• average concentrations
• kinetic chemical reaction rates
• singlet-triplet dynamics

Raster image correlation spectroscopy (RICS)

As described above, FCS measures the fluctuations in fluorescence at a single point on a sample. However, measuring molecular movement in live cells over a larger area while maintaining high sensitivity is more demanding. Raster image correlation spectroscopy (RICS) exploits the time-related information that exists in sequential laser-scanned images to correlate fluorescence intensity fluctuations over time on a pixel-by-pixel basis. Data from fluctuations in fluorescence intensity can be analysed as a function of time and space by autocorrelation mathematics to reveal information on diffusion, binding, flow and the state of molecular aggregation. RICS is able to provide highly detailed positional and time-resolved data on the movement of the labeled molecules in the microsecond to second range with single molecule sensitivity. Importantly, RICS can reveal fast molecular movement by filtering out the slower bulk cell movement, which is invaluable as many cellular components are constantly in motion. RICS software has been designed to be suitable for use on data from most commercially available confocal laser scanning microscopes.