Fluorescence correlation spectroscopy (FCS) is increasingly being used to assess the movement of particles diffusing in complex, optically dense surroundings, in which case measurement conditions may complicate data interpretation. It is considered how a single-photon FCS measurement can be affected if the sample properties result in scattering of the incident light. FCS autocorrelation functions of Atto 488 dye molecules diffusing in solutions of polystyrene beads are measured, which acted as scatterers. Data indicated that a scattering-linked increase in the illuminated volume, as much as two fold, resulted in minimal increase in diffusivity. To analyze the illuminated beam profile, Monte-Carlo simulations were employed, which indicated a larger broadening of the beam along the axial than the radial directions, and a reduction of the incident intensity at the focal point. The broadening of the volume in the axial direction has only negligible effect on the measured diffusion time, since intensity fluctuations due to diffusion events in the radial direction are dominant in FCS measurements. Collectively, results indicate that multiple scattering does not result in FCS measurement artifacts and thus, when sufficient signal intensity is attainable, single-photon FCS can be a useful technique for measuring probe diffusivity in optically dense media.
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