Using reflectometry to minimize the dependence of fluorescence intensity on optical absorption and scattering

The total diffuse reflectance R( T ) and the effective attenuation coefficient µ( eff ) of an optically diffuse medium map uniquely onto its absorption and reduced scattering coefficients. Using this premise, we developed a methodology where R( T ) and the slope of the logarithmic spatially resolved reflectance, a quantity related to µ( eff ), are the inputs of a look-up table to correct the dependence of fluorescent signals on the media’s optical properties. This methodology does not require an estimation of the medium’s optical property, avoiding elaborate simulations and their errors to offer accurate and fast corrections. The experimental demonstration of our method yielded a mean relative error in fluorophore concentrations of less than 4% over a wide range of optical property variations. We discuss how the method developed can be employed to improve image fidelity and fluorochrome quantification in fluorescence molecular imaging clinical applications.