The fluorescence spectrum measured from a fluorophore in tissue is affected by the absorption and scattering properties of the tissue, as well as by the measurement geometry. We analyze this effect with Monte Carlo simulations and by measurements on phantoms. The spectral changes can be used to estimate the depth of a fluorescent lesion embedded in the tissue by measurement of the fluorescence signal in different wavelength bands. By taking the ratio between the signals at two wavelengths, we show that it is possible to determine the depth of the lesion. Simulations were performed and validated by measurements on a phantom in the wavelength range 815-930 nm. The depth of a fluorescing layer could be determined with 0.6-mm accuracy down to at least a depth of 10 mm. Monte Carlo simulations were also performed for different tissue types of various composition. The results indicate that depth estimation of a lesion should be possible with 2-3-mm accuracy, with no assumptions made about the optical properties, for a wide range of tissues.

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We present a novel method for estimating the depth of a fluorescent lesion in tissue based on measurements of the fluorescence signal in different wavelength bands. The measured fluorescence spectrum following irradiation by excitation light at the surface is a function of several parameters, because the fluorescence light has to pass through tissue with characteristic scattering and absorption properties. Thus, the intrinsic fluorescence spectrum will be altered, in a way determined by the tissue optical properties, the depth of the fluorophore, and also by the geometry of the light irradiation and the detection system. By analyzing the ratio between the signals at two wavelengths we show that it is possible to estimate the depth of the lesion. We have performed Monte Carlo simulations and measurements on an Intralipid phantom in the wavelength range 850 -1000 nm. By taking the ratio between the signals at the wavelengths 875 and 930 nm the depth of a fluorescing layer could be determined with 0.8 mm accuracy down at least a depth of 10 mm. Monte Carlo simulations were also performed for different tissue types with various composition. The results indicate that depth estimation of a lesion is possible with no assumptions made about the optical properties for a wide range of tissues.

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