Confocal fluorescence polarization microscopy in turbid media: effects of scattering-induced depolarization

We present an experimental and theoretical study of confocal fluorescence polarization microscopy in turbid media. We have performed an experimental study using a fluorophore-embedded polymer rod immersed in aqueous suspensions of 0.1 and 0.5 microm diameter polystyrene microspheres. A Monte Carlo approach to simulate confocal fluorescence polarization imaging in scattering media is also presented. It incorporates a detailed model of polarized fluorescence generation that includes sampling of elliptical polarization, excited-state molecular rotational Brownian motion, and dipole fluorescence emission. Using both approaches, we determine the effects of the number of scattering events, target depth, photon scattering statistics, objective numerical aperture, and pinhole size on confocal anisotropy imaging. From this detailed analysis and comparison of experiment with simulation, we determine that fluorescence polarization is maintained to depths at which meaningful intensity images can be acquired.