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Optical methods in medicine are exploit the light propagation through the biological tissue, to non-invasively extract morphological and geometrical parameters of the tissue under investigation, from the detected light. However, because the transport of photons into biological tissues of interest is weakened due to losses caused by multiple light scattering, absorption and reflectance, the depth resolution and photon penetration depth of these optical methods still need to be improved. The optical properties of biological tissues can be controlled using special biocompatible hyperosmotic agents or Optical Clearing Agents (OCA). The latter might penetrate inside a tissue, interact with and modify its natural high-scattering formation through different ways and thus, increase the photons penetration within the biological tissue. The aim of this work was to study the modifications in depth resolution of the proposed Spatially Resolved Multi-Modal Spectroscopy (SRMMS) method by combining it with an optical clearing approach and to estimate the applicability of this method for future clinical use.
As an OCA, PEG-400/DMSO (80%/20% vol/vol) and Sucrose/PEG-400/PG (50%/45%/5% vol/vol) solutions were used. Depending on the experimental condition, either one of 2 OCA, or saline solution or nothing ("dry" condition) was applied on the skin surface of 2-layer hybrid model, made of skin layer on top and the gel layer, containing exogenous fluorophore Chlorin e6, on bot.
The SRMMS probe made of one central excitation fiber and 4 rings of collecting fibers for collecting spectroscopic data with the separation by depth, was placed in contact with the skin at the area of application. Then, Diffuse Reflectance and excited on a different wavelengths (365,385,395, 405 and 415 nm) Autofluorescence spectra were aquired at initial time point, and then every 4 minutes within 36 minutes. Aquired DR and AF spectra were preprocessed, and the Area Under the Curve (AUC) values were calculated for the specific wavelength bandwidths of interest for the skin-AF and Ce6-fluorescence spectra obtained at every of the 5 excitation peaks. AUC values were normalized to the T0 respective values to get the time kinetics curves of the spectroscopic signals.
Time kinetics of skin-AF and Ce6-fluorescence spectrum AUC showed that under 36 minutes of OCA application it is possible to observe certain enhanced light penetration effect through an increasing of normalized Ce6 AUC kinetics and a decreasing of normalized AF AUC kinetics. Thus, it was possible to observe that optical clearing led to increased depth resolution with time. Moreover, the effect of clearing itself was observed to be dependent on the chosen OCA, while the saline solution application didn’t cause significant changes in normalized fluorescence intensities. The result of “dry” condition experiment highlighted that while there were no significant changes in AF spectrum amplitudes, a strong increase of Ce6 fluorescence emission intensity was collected from the bottom gel layer. This is probably due to drying of an ex vivo skin sample and the applied probe pressure effect.
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