Fluorescence and diffuse reflectance provide similar accuracy in recovering fluorophore concentration at short source-detector separations.

Quantitative fluorescence spectroscopy requires corresponding reflectance measurements to correct for tissue absorption and scattering. However, it is unclear whether fluorescence adds value beyond the diffuse reflectance measurements necessary for correction. The goal of this study was to compare the accuracy of fluorescence and diffuse reflectance spectroscopy in recovering the concentration of a high-extinction fluorophore, methylene blue (MB), using a compact fiber-optic probe. Fluorescence and diffuse reflectance measurements of tissue simulating phantoms were made using a fiber-optic probe with source-detector separations of 288-1300 μm. Average error in recovered fluorophore concentration was 20.4% for fluorescence and 15.0% for reflectance, though this difference was not significant (p=0.77). Both methods returned concentrations that were similar to known MB concentrations (p≥0.79 in both cases). Fluorescence quantification of the concentration of a high extinction fluorophore did not significantly improve accuracy relative to diffuse reflectance. Investigators should consider whether fluorescence measurements are necessary for a given application.