We used a fiberoptic probe connected to a spectrometer to measure the response of brain tissue excited with blue light (wavelength: λ = 405 nm) followed by interrogation with broadband white light (λ = 450–720 nm) delivered through the fiberoptic probe tip to compensate for signal losses due to light absorption and scattering by the tissue using novel light-transport modeling methods.7 (link) The relative contributions of oxyhemoglobin and deoxyhemoglobin to the total light absorption were determined in situ by using the measured white-light reflectance spectrum fitted to a diffusion model of the reflectance where the intrinsic absorption spectra of each of these proteins were assumed to be known.
A mathematical model7 (link) (Appendix) was then used to quantify the absolute concentration of PpIX based on the measured fluorescence spectrum. Since the fluorescence measurements are distorted by variations in tissue optical properties, quantitative determination of the concentration of fluorescing molecules requires knowledge of the absorption and scattering spectra of the tissue being sampled. Moreover, PpIX is not the only molecule contributing to the measured fluorescence spectrum (other contributors include tissue autofluorescence and photoproducts such as photoprotoporphyrin resulting from PpIX photobleaching3 (link)). However, the spectral shapes of the primary fluorophores of interest are known, which allows spectral decomposition to be used to determine their relative contributions, and in the case of PpIX, the absolute concentration (CPpIX). By incorporating these light-transport modeling methods that eliminate the distorting effects of variations in tissue optical properties and account for the presence of multiple fluorescent species, quantitative measurements of CPpIX can be obtained intraoperatively and in vivo with an unprecedented degree of sensitivity and fidelity.