Determine scattering coefficient and anisotropy of scattering of murine tissues using reflectance-mode confocal microscopy

Different techniques have been developed to determine the optical properties of turbid media, which include collimated transmission, diffuse reflectance, adding-doubling and goniometry. While goniometry can be used to determine the anisotropy of scattering (g), other techniques are used to measure the absorption coefficient and reduced scattering coefficient (μ_s(1-g)). But separating scattering coefficient (μ_s) and anisotropy of scattering from reduced scattering coefficient has been tricky. We developed an algorithm to determine anisotropy of scattering from the depth dependent decay of reflectance-mode confocal scanning laser microscopy (rCSLM) data. This report presents the testing of the algorithm on tissue phantoms with different anisotropies (g = 0.127 to 0.868, at 488 nm wavelength). Tissue phantoms were made from polystyrene microspheres (6 sizes 0.1-0.5 μm dia.) dispersed in both aqueous solutions and agarose gels. Three dimensional images were captured. The rCSLM-signal followed an exponential decay as a function of depth of the focal volume, R(z)ρexp(-μz) where ρ (dimensionless, ρ = 1 for a mirror) is the local reflectivity and μ [cm-1] is the exponential decay constant. The theory was developed to uniquely map the experimentally determined μ and ρ into the optical scattering properties μ_s and g. The values of μ_s and g depend on the composition and microstructure of tissues, and allow characterization of a tissue.