Assessment of optical clearing agents using reflectance-mode confocal scanning laser microscopy

The mechanism of action of clearing agents to improve optical imaging of mouse skin during reflectance-mode confocal microscopy was tested. The dermal side of excised dorsal mouse skin was exposed for one hour to saline, glycerin, or 80% DMSO, then the clearing agent was removed and the dermis placed against a glass cover slip through which a confocal microscope measured reflectance at 488 nm wavelength. An untreated control was also measured. The axial attenuation of reflectance signal, R(z_f) versus increasing depth of focus z _f behaved as R = ρexp(-μz_f2G), where ρ is tissue reflectivity and μ is attenuation [cm^-1]. The factor 2G accounts for the in/out path of photons, and the numerical aperture of the lens. The ρ, μ data were mapped to values of scattering coefficient (μ_s [cm^-1]) and anisotropy of scattering (g). Images showed that glycerin significantly increased the g of dermis from about 0.7 to about 0.99, with little change in the μ_s of dermis at about 300 cm^-1. DMSO and saline had only slight and inconsistent effects on g and μ_s.