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Fluorescence fluctuation spectroscopy infers molecular properties from variations in detected fluorescence, rather than only from its time-averaged intensity. Diffusion of fluorescent molecules through a small illuminated region changes their number in that region; binding or changes of fluorescent state can change their brightness. Fluorescence correlation spectroscopy studies the time dependence of these fluctuations through an autocorrelation function.^[ref]

For a stationary recorded intensity $I(t)$, put $\delta I(t)=I(t)-⟨I⟩$. A common normalized convention isFor an ideal uniformly weighted observation volume with independent equally bright particles, a Poisson distribution for the particle number gives $G(0)=1/\overline{N}$, excluding the detector's instantaneous shot-noise contribution. The same mean signal could therefore arise from many dim particles or fewer bright ones, while their relative fluctuations differ. Real confocal profiles introduce an effective observation volume and model-dependent shape factors.

A confocal implementation uses a focused laser, spatial filtering of collected fluorescence and photon-sensitive detection to record an intensity trace. Faster diffusion usually shortens the correlation time. Binding kinetics, background, bleaching and detector effects can also modify the trace, so a fitted timescale needs a physical model.^[ref]

The 1972 experiment by Douglas Magde and colleagues measured equilibrium fluctuations during binding of ethidium bromide to DNA. The measured correlations revealed both diffusion coefficients and chemical rate constants. It showed how apparently unwanted fluctuations can expose molecular dynamics without imposing a macroscopic concentration jump.^[ref]

Yan Chen is the first author of the 1999 photon-counting-histogram study in fluorescence fluctuation spectroscopy. The analysis used the distribution of detected photon counts in short intervals to estimate molecular brightness and the average number of fluorescent molecules in the observation volume.^[ref]

Two samples can have the same mean fluorescence while containing different numbers of molecules with different individual brightnesses. Their count distributions can help distinguish these possibilities when interpreted with the instrument's illumination profile and counting statistics. This entry identifies the researcher through that paper's byline and affiliation, without conflating other scientists named Yan Chen.