= Fluorescence fluctuation spectroscopy
<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>.<Fluorescence Correlation Spectroscopy by the MIT So Lab>[Overview and autocorrelation explanation]{ref}
For a stationary recorded intensity $I(t)$, put $\delta I(t)=I(t)-\langle I\rangle$. A common normalized convention is
$$
G(\tau)=\frac{\langle\delta I(t)\delta I(t+\tau)\rangle}{\langle I\rangle^2}.
$$
For 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.<Fluorescence Correlation Spectroscopy by the MIT So Lab>[Detection-volume geometry, intensity fluctuations and limitations]{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.<Thermodynamic Fluctuations in a Reacting System—Measurement by Fluorescence Correlation Spectroscopy by Douglas Magde and others>[Abstract, equilibrium reaction-product fluorescence and DNA-binding experiment]{ref}
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