Quantitative fluorescence measurements from tissue using confocal detection

Fluorescence intensity measurements from fluorophore molecules present in tissue can be affected by the intrinsic scattering and absorption of the tissue. Using detection of the fluorescence from small regions of tissue the fluorescent intensity signal is not significantly affected by the background absorption in the tissue. Confocal microscopic measurements have been examined here in an effort to develop a system in which the fluorescent intensity measured from tissue is linearly proportional to only the fluorophore concentration and the input light irradiance. Monte Carlo simulations confirm that by constraining the detected area to smaller than the average scattering length, that the fluorescence measurements are not significantly affected by intrinsic absorption. This type of system is useful for quantification of photosensitizer concentrations in tissue during photodynamic therapy or for pharmacokinetic measurements of uptake in tissue. Preliminary measurements confirm that this method should be equivalent to fluorescence-based tissue extraction methods of photosensitizer uptake in tissue.