Fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) analyzes temporal fluctuations in fluorescence from a tiny observation volume to estimate molecular concentrations, diffusion, and interactions.

Autocorrelation function: A function measuring how strongly a signal correlates with a time-shifted copy of itself. FCS extracts molecular timescales by correlating fluorescence intensity fluctuations across time delays.
Fluorescence cross-correlation spectroscopy: A two-color fluctuation method that detects correlated motion of differently labeled molecular species. Cross-correlating channels lets FCS test whether distinct labeled molecules move together.
Fluorescence: Light emission from a substance after it absorbs photons and enters an excited electronic state. FCS detects emitted fluorescence as labeled molecules fluctuate through the observation volume.
Fluorescence intensity distribution analysis: A fluorescence fluctuation method that analyzes the distribution of intensity values to estimate molecular brightness and number. FIDA uses intensity distributions, whereas conventional FCS emphasizes temporal correlations.
Brownian motion: Random motion of particles caused by collisions with molecules in their surrounding medium. Diffusing fluorescent molecules enter and leave the observation volume through Brownian motion.
Molecular diffusion: The net movement of molecules from regions of higher concentration toward lower concentration through random motion. FCS estimates diffusion coefficients from the characteristic time molecules spend in its observation volume.
Fluorophore: A molecule or molecular group that absorbs light and emits fluorescence. A fluorophore supplies the detectable signal whose fluctuations FCS analyzes.
Number and brightness analysis: A fluorescence fluctuation method that estimates particle number and molecular brightness from pixel-wise intensity variance. N&B maps fluctuation statistics across image pixels rather than relying on a single point-volume correlation.
Confocal microscopy: An optical imaging method that rejects out-of-focus light using a spatial pinhole. Confocal optics help define the small volume whose fluorescence fluctuations FCS measures.
Molecular binding: A molecular association in which two or more molecules form a complex through noncovalent or covalent interactions. Binding can change diffusion and fluorescence fluctuations, allowing FCS to probe molecular interactions.

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