Progress in photodynamic therapy (PDT) requires development of novel protocols. We report on performance of PDT employing chlorine-based photosensitizers and irradiation at wavelengths of 405 and 660 nm with monitoring by fluorescence imaging (FI) and optical coherence tomography (OCT). The study includes numerical simulations, model and animal experiments, as well as clinical monitoring.

Photodynamic therapy (PDT) is a modern treatment technique employed as an antitumor, antibacterial, or rejuvenation aid in superficial tissues. The use of fluorescent photosensitizers (PSs) implements the principles of the theranostics when the applied medical agents serve both for diagnostic and treatment purposes. For efficient PDT performance it is important to evaluate the in-depth distribution of PSs in tissues prior to irradiation. Fluorescence imaging is a common technique to monitor the distribution of PSs in tissue. However, in-depth resolution is challenging. Chlorin-based PSs reveal two narrow fluorescence excitation peaks at 405 and 660nm providing additional diagnostic opportunities. We demonstrate that the ratio of the fluorescence signals upon excitation at these wavelengths provides the evaluation of the PS penetration depth after topical application. The study is based on Monte Carlo simulations that are in agreement with phantom experiments. The effect of medium optical properties on the depth-dependent fluorescence signal ratio is analyzed.

The efficiency of light scattering by nanoparticles formed using the method of picosecond laser ablation of silicon in water and by nanoparticles of mechanically grinded mesoporous silicon is compared. The ensembles of particles of both types possess the scattering coefficients sufficient to use them as contrast agents in optical coherence tomography (OCT), particularly in the range of wavelengths 700-1000 nm, where the absorption of both silicon and most biological and mimicking tissues is small. According to the Mie theory the main contribution to the scattering in this case is made by the particles having a relatively large size (150-300 nm). In the experiments on visualising the agar phantom surface by means of OCT, the contrast of the medium boundary, provided by nanoparticles amounted to 14 dB and 30 dB for the ablated particles and the porous silicon powder, respectively. The numerical simulation of OCT images of skin in the presence of nanoparticles, confirmed the efficiency of using them as a contrast agent.

The aim of the study is to develop approaches for fluorescence monitoring and planning of photodynamic therapy employing chlorine series photosensitizers. Materials and Methods. The study included numerical simulations and experiments with optical agar phantoms of biotissue and human skin in vivo. Fluorescence imaging was used as a method of optical monitoring. Chlorine series photosensitizer Photoditazin (Veta Grand, Russia) was employed. Numerical simulation of light propagation was performed with Monte-Carlo technique for a multilayer skin model. Results. It was demonstrated that in the case of two-wavelength fluorescence monitoring of photosensitizer penetration into the tissue the ratio of fluorescence signals excited at wavelengths of 405 and 660 nm can be used as a characteristic of photosensitizer penetration depth in biological tissue. The results of numerical simulations are in good agreement with the results of model experiments on agar phantoms and pilot in vivo experiment. Radiant exposure and absorbed light dose maps at the wavelengths of 405 and 660 nm were calculated employing Monte-Carlo technique; the dependencies of the characteristic dose values on the optical properties of the medium were analyzed. Conclusion. Two-wave fluorescence imaging technique allows for non-invasive estimation of chlorine series photosensitizer penetration depth into the biotissue after topical application, while numerical simulation by Monte-Carlo method allows for more accurate choice of the light exposure dose for photodynamic therapy depending on optical properties of the tissue and the radiation wavelength.

Diffuse optical spectroscopy (DOS) and its modification employing structured illumination are widely used in monitoring biotissue oxygenation. In such measurements it is important to know the probing volume for definite source-detector configuration; however, it cannot be measured directly. Monte Carlo simulations allow to trace the probing depth of each individual photon contributing to the signal, which provides a numerical solution for this problem. In this study we investigate distributions of photons over maximal depth reached in turbid media (probing depth) with optical parameters typical for cutaneous tissues at the wavelength of 600 nm. Different configurations of probing illumination are considered, such as collimated point source, one-dimension sinusoidal and rectangular patterns. For collimated point source and zero source-detector separation the number of collected photons monotonously decreases with the probing depth while a pronounced maximum in the distribution is manifested with the increase of source-detector separation. The position of this maximum shifts to higher depths with the decrease of µa. For one-direction sinusoidal and rectangular illumination patterns it is shown that when the photons are collected near the center of a bright stripe, the peak of the distribution remains close to the surface. When the photons are collected near the center of a dark stripe the peak shifts towards higher depths with the decrease in spatial duty cycle and spatial frequency of the illumination pattern. Employment of rectangular illumination pattern seems more efficient for DOS applications due to wider abilities for controlling probing depth.

In this work we studied the efficiency of spatially modulated illumination in optical diffuse reflectometry (ODR) and analyzed various probing patterns. The optimal illumination pattern was determined from the series of Monte Carlo numerical experiments on structured illumination and comparison of the parameters of fluence distribution within tissue. We considered the following illumination profiles: sinusoidal patterns with different spatial frequencies k (1 - 2.5 mm-1); piecewise constant patterns with the fixed duty cycle w = 2 and various strip width (0.2 – 1 mm); piecewise constant patterns with the fixed strip width (0.1 mm) and various duty cycle (3 – 11). Assuming the same total incident intensity for all patterns, we observed the growth in intensity at depth with decreasing value of spatial frequency for sinusoidal pattern, the similar tendency of intensity distribution was for piecewise constant patterns with the fixed strip width (or duty cycle) and duty cycle increase (or increase of a strip width, respectively). The intensity distributions within the sample are almost identical for sinusoidal and piecewise constant patterns with matching peaks and lows. However, probing by piecewise constant profile of illumination provide more local information about sample’s properties. A prototype of an ODR system for probing with the structured illumination was designed. The system consists of PC-controlled optical projection system, polarization filters and a CCD camera. The approbation of the system was carried out on the optical phantoms with optical properties close to those of biological tissues and on skin of human volunteers.

Optical phantoms mimicking optical properties of real biotissues in the visible and IR spectral regions are developed based on measurements of the spectral characteristics of ex vivo samples of laboratory mouse biotissues. The phantoms are composed of aqueous solutions of Lipofundin, Indian ink and red ink with different spectral characteristics. The deviations of the measured absorption and scattering coefficients of phantoms in the wavelength range 480-580 nm from the corresponding values for real biotissues do not exceed 25 % and 2 %, respectively. For phantoms in the wavelength region 580-880 nm, the deviations of the absorption coefficient do not exceed 40 % and the deviations of the scattering coefficient do not exceed 25 %. These values, in general, fall within the range of variations for different individual mice of one strain.