FCS measurements require a confocal microscope fitted with an objective with a high numerical aperture with the basic microscope setup summarized in (a). The use of a pinhole creates a Gaussian-shaped detection volume, approximately 0.25–0.5 fl in diameter, and encompassing a plasma membrane region of ∼0.3 μm2 (b). As fluorescent particles pass through the detection volume, they produce time-dependent fluctuations in fluorescence intensity (c). The amplitude of these fluctuations (δI) can be compared with that of the mean fluorescence intensity (⟨I⟩) at time point t with that of a fluctuation at a later time point (t + τ). Analysis of an ensemble of τ values allows the autocorrelation function (Gτ) to be determined (d) and the average dwell time (τD) of the fluorescent species can be derived from the midpoint decay of the autocorrelation curve. A single time-correlated autocorrelation trace can contain multiple components (e.g. τD1, τD2 etc.; e) that represent the different molecular complexes present within the confocal volume distinguished by their different rates of diffusion (typical time scales stated).