Measuring tissue optical properties in vivo using reflectance-mode confocal microscopy and OCT

The ability to separately measure the scattering coefficient (μ_s [cm^-1]) and the anisotropy (g) is difficult, especially when measuring an in vivo site that can not be excised for bench-top measurements. The scattering properties (μ_s and g) can characterize the ultrastructure of a biological tissue (nuclear size, mitochondra, cytoskeletion, collagen fibers, density of membranes) without needing an added contrast agent. This report describes the use of reflectance-mode confocal scanning laser microscopy (rCSLM) to measure optical properties. rCSLM is the same as optical coherence tomography (OCT) when the OCT is conducted in focus-tracking mode. The experimental measurement involves translating the depth of focus, Z_f, of an objective lens, down into a tissue. As depth z increases, the reflected signal R decreases due to attenuation by the tissue scattering (and absorption, μ_a). The experimental data behaves as a simple exponential, R(z) = ρ exp(-μz _f) where ρ is the local reflectivity (dimensionless) and μ [cm^-1] is an attenuation coefficient. The relationship between (ρ,μ) and (μ_s,g) is: μ = (μ_s a(g) + μ_a)2 G(g,NA) ρ-μ_s L_f b(g,NA) where a(g) is a factor that drops from 1 to 0 as g increases from 0 to 1 (determined by Monte Carlo simulations) allowing photons to reach the focus despite scattering, G is a geometry factor describing the average photon pathlength that depends on the numerical aperture (NA) of the lens and the anisotropy (g), L_f is the axial extent of the focus, and b(g,NA) is the fraction of scattered light that backscatters into the lens for detection.