Calibration Of Fluorescence Reflectance Reference Phantoms

Fluorescence intensity is often standardized by comparing the unknown sample signal to that of a reference solution with a known concentration of a reference fluorophore. To use this technique for in vivo fluorescence reflectance measurements standardization, a reference sample that also mimics the scattering and absorption properties of the tissue would need to be used. A simpler approach to fluorescence reflectance measurements standardization is to express the intensity of the measured fluorescence as a ratio of the excitation irradiance to the fluorescence radiance. This ratio of radiometric quantities can be measured by normalizing the measured fluorescence image to a reflectance image acquired with a reflection standard at the excitation wavelength (without the emission filter). Instruments could be calibrated to report their intensity results using this dimensionless ratio. The calibration could also be transferred from a reference calibrated instrument to other instruments through the use of fluorescence phantoms. Reporting fluorescence intensity measurements using this dimensionless ratio will ease instrument standardization and comparison of fluorescence reflectance results between instruments, vendors and applications.