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

The ability to separately measure the scattering coefficient (μs [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 (μs 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, zf, 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, μa). The experimental data behaves as a simple exponential, R(z) = ρ exp(-μzf) where ρ is the local reflectivity (dimensionless) and μ [cm⁻¹] is an attenuation coefficient. The relationship between (ρ,μ) and (μs,g) is: μ = (μs a(g) + μa) 2 G(g,NA) ρ = μs Lf 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), Lf is the axial extent of the focus, and b(g,NA) is the fraction of scattered light that backscatters into the lens for detection.