Fluorescence imaging modalities are currently a routine tool for the assessment of marker distribution within biological tissues, including monitoring of fluorescent photosensitizers (PSs) in photodynamic therapy (PDT). Conventional fluorescence imaging techniques provide en-face two-dimensional images, while depth-resolved techniques require complicated tomographic modalities. In this paper, we report on a cost-effective approach for the estimation of fluorophore localization depth based on dual-wavelength probing. Owing to significant difference in optical properties of superficial biotissues for red and blue ranges of optical spectra, simultaneous detection of fluorescence excited at different wavelengths provides complementary information from different measurement volumes. Here, we report analytical and numerical models of the dual-wavelength fluorescence imaging of PS-containing biotissues considering topical and intravenous PS administration, and demonstrate the feasibility of this approach for evaluation of the PS localization depth based on the fluorescence signal ratio. The results of analytical and numerical simulations, as well as phantom experiments, were translated to the in vivo imaging to interpret experimental observations in animal experiments, human volunteers, and clinical studies. The proposed approach allowed us to estimate typical accumulation depths of PS localization which are consistent with the morphologically expected values for both topical PS administration and intravenous injection.

An approach to fabricating agar phantoms mimicking spectral optical properties of biological tissues with fluorescent inclusions is proposed, which allows one to imitate the problem of optical visualisation of superficial biological tissues after the administration of a chlorin-based photosensitiser. The different arrangement of a fluorescent layer within a phantom makes it possible to simulate biological tissue in the cases of both topical application and intravenous injection of a photosensitiser. It is shown that absorption and scattering spectra of phantoms are in good agreement with the spectra of real biological tissues in the wavelength range of 500-800 nm. Changes in spectra of absorption and scattering coefficients of phantoms, as well as in their fluorescent properties induced by the addition of a fluorescent marker (chlorinbased photosensitiser) are demonstrated.

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