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The motivation for the present study was the need to develop methods of urgent intraoperative biopsy during surgery for removal of intracranial tumors. Based on the experience of previous joint work of GPI RAS and N.N. Burdenko National Medical Research Center of Neurosurgery to introduce fluorescence spectroscopy methods into clinical practice, an approach combining various optical-spectral techniques, such as autofluorescence spectroscopy, fluorescence of 5-ALA induced protoporphyrin IX, diffuse reflection of broadband light, which can be used to determine hemoglobin concentration in tissues and their optical density, Raman spectroscopy, which is a spectroscopic method that allows detection of various molecules in tissues by vibrations of individual characteristic molecular bonds. Such a variety of optical and spectral characteristics makes it difficult for the surgeon to analyze them directly during surgery, as it is usually realized in the case of fluorescence methods – tumor tissue can be distinguished from normal with a certain degree of certainty by fluorescence intensity exceeding a threshold value. In case the number of parameters exceeds a couple of dozens, it is necessary to use machine learning algorithms to build a intraoperative decision support system for the surgeon. This paper presents research in this direction. Our earlier statistical analysis of the optical-spectral features allowed identifying statistically significant spectral ranges for analysis of diagnostically important tissue components. Studies of dimensionality reduction techniques of the optical-spectral feature vector and methods of clustering of the studied samples also allowed us to approach the implementation of the automatic classification method. Importantly, the classification task can be used in two applications – to differentiate between different tumors and to differentiate between different parts of the same (center, perifocal zone, normal) tumor. This paper presents the results of our research in the first direction. We investigated the combination of several methods and showed the possibility of differentiating glial and meningeal tumors based on the proposed optical-spectral analysis method.

The purpose of the work is to follow the dynamics of changes in fluorescent signals in the near-surface layers of tissue of injured areas of the back of laboratory animals, which will allow, by indirect evidence, to evaluate the information content of fluorescence diagnosis for subsequent possible diagnostic monitoring of photodynamic therapy of the spinal cord. The model animals were Wistar rats. Two types of contusions were modeled: pneumo-contusion and contusion by a falling load. Methylene blue and indocyanine green were used as photosensitizers. Fluorescence measurements were carried out by imaging and spectrometric methods. A stroboscopic fluorescence imager with an excitation wavelength of 630 nm was used to acquire fluorescence images. The LESA-01-BIOSPEC spectrometer with a He-Ne laser excitation allowed to obtain spectra. It was shown that both methods make it possible to estimate the fluorescence value of methylene blue and indocyanine green in the tissues under study. Moreover, the photographic method also allows to obtain the spatial distribution of fluorescence. The general trend found in the data is a more intense and uniform fluorescence of the dorsal region of rats with methylene blue and a less intense, but more contrasting distribution of indocyanine green. The presented methods are non-invasive, which makes them attractive for diagnostic use. However, due to the shallow depth of signal reception, the condition of the spine can be determined only indirectly, by the condition of the near-surface layers of tissue that accumulate the photosensitizer.

Targeted therapy and immunotherapy are considered promising novel therapies capable of increasing the effcacy of prostate cancer (PCa) treatment. The purpose of the study was to obtain and characterize TRAMP-C2 subcutaneous and ascite-solid models of prostate cancer in C57Bl/6j mice to study specifc anti-tumor activity of the candidate molecules of targeted drugs and adjust immunotherapy strategies in an evidence-based manner. Material and Methods. We used cultured TRAMP-C2 cells in subcutaneous and ascites mouse prostate cancer models. Histological and immunohistochemical methods were used to study the tumor tissues. Results. The high in vivo growth ability of TRAMP-C2 cells was demonstrated in subcutaneous and intraperitoneal inoculation of C57Bl/6j mice. These tumors are characterized by a high reproducibility and level of PSMA expression. Histological study showed that subcutaneous and carcinomatous TRAMP-C2 tumor nodules had solid structure morphologically corresponding to low differentiated neoplasm, cytomorphological analysis of smears showed that peritoneal fuid containd a large number of rounded tumor cells, macrophages and erythrocytes. Conclusion. The obtained subcutaneous and ascite-solid models of TRAMP-C2 can be useful for the development of new ways to effectively treat cancer, including targeted and immunotherapy, as well as for the experimental study of biotherapeutic effects using PSMA as a target, and photoinduced effects.

Methylene blue (MB) is a promising photosensitizer (PS) for the treatment of pathological neoplasms, since it has both photodynamic activity (under laser irradiation) and redox and catalytic properties (in the absence of light). In the framework of this work, using spectroscopic methods, the effect of intravenous administration of MB on tissue oxygenation of hemoglobin in small animals in vivo in tumor and normal tissues was analyzed. The influence of MB on cell metabolism was analyzed. It has been shown that the use of MB promotes an increase in oxygen consumption by the tumor, and also leads to a shift in metabolism towards oxidative phosphorylation. It was shown that the use of MB contributes to an increase in oxygen consumption by the tumor, and also leads to a shift in metabolism towards oxidative phosphorylation.

A wide range of literature sources report on the potential benefits of transdermal drug delivery. Among these advantages, the following are distinguished – minimal injury, reduction of side effects, and prevention of degradation or metabolism in the gastrointestinal tract or liver. However, transdermal delivery of most molecules often excludes due to the barrier function of the skin, which prevents the penetration of exogenous substances. To overcome this barrier and increase skin absorption, ethosomal complexes use, by means penetration into the deep layers of the skin and/or systemic circulation is possible. This work devotes to the development of a non-invasive method for assessing the depth of penetration by ethosomes with methylene blue (MB) into the skin during application and photodynamic exposure. MB as photosensitizer (PS) was chosen, since there are a sufficient number of publications on its positive effect on the restoration of the cell’s respiratory chain of various organs and therefore the restoration of their metabolism. Besides MB has proven to be an effective PS, destructed pathogenic microbes and viruses, including SARS-CoV-2. However, for more effective Covid-19 therapy and antibiotic-resistant microbial diseases, the penetration of MB into the vascular system of the epidermis or mucous tissue is required. Nowadays, the existing methods for assessing the penetration depth of PS are time consuming and require the use of animal skin or model samples. The LESA-01 BIOSPEC system with specially designed optical adapters that allow assessing the drug fluorescence intensity on skin surface and at a depth of up to 2 mm in the investigation was used.

Introduction. Today, antimicrobial photodynamic therapy (a-PDT) becomes more and more popular, because of it pronounced bactericidal activity, anti-inflammatory effect. At the same time, no animal model studies have been conducted on morphological changes in cells after exposure to PDT on venous ulcers (VU) when using different types of photosensitizers (PS). The problem of comparing morphological changes in tissues when using a-PDT vs conventional PDT have not yet been resolved.Aim. Evaluation of the effectiveness of PDT and APDT in a comprehensive examination of trophic ulcers in an in vivo experiment and compare them with standard methods for the treatment of VLU.Materials and мethods. A series of experiments was conducted on 21 rabbits, separated into 3 equal groups of 7 rabbits each. Venous trophic ulcer was originally modeled for all rabbits. To obtain a VU, we performed an additional ligation of v. femoralis. The control group received standard therapy for VU. The PDT group had PDT with Photosens. The a-PDT group underwent a-PDT using Cholosens. Every 3 days, picture of local inflammation, regeneration rate and ulcer volume were determined. A morphological study of VLU was carried out on the first, 9th and 15th days.Results. The a-PDT group, day 15: 100% wound epithelization. Control group, day 21st: The volume of wounds decreased on average by 50%. The PDT group: 100% wound epithelization. The morphological study indicated a positive trend in the a-PDT group compared with PDT and control groups, which resulted in a decrease in the total mass of necrotic detritus, a change in the quantitative and qualitative composition of inflammatory infiltrate.Conclusion. The data obtained indicate that the use of a-PDT is recommended in treatment of VLU. Both PDT and a-PDT methods showed better results in comparison with standard therapy.

Indocyanine green (ICG), when in free form in a liquid, can form stable nanoparticle structures or colloidal solution, while changing its spectroscopic properties. In the work, the aggregation degree and the average size of nanoparticles depending on the concentration of a colloidal solution of indocyanine green (ICG NPs) in the form of J-aggregates were investigated by various methods based on light scattering. The size of nanoparticles is an important parameter from the point of view of clinical application, because the technique of intravenous administration of drugs, in order to avoid microvascular thrombosis and embolism, provides dosage forms with inclusions of individual molecules or their clusters, not exceeding 500 nm diameter. In turn, small nanoparticles less than 30 nm lead to prolonged circulation of the drug in the body with an increased possibility of permeation into cells of healthy tissue. In the course of studies, it was found that an increase in the concentration of ICG NPs in the solution leads to an increase in the average size of spontaneously formed J-aggregates, which, in turn, leads to a decrease in the absorption coefficient in the aggregates. Presumably, this phenomenon, i.e. the established nonlinear dependence of the J-aggregate absorption on its size, can be explained by the formation of absorption centers on the J-aggregate surface in the form of mobile surface molecules. The threshold range of ICG molecule concentration was determined, at which there is a transition from aggregation with an increase in size with a slow addition of ICG J-aggregate molecules in height to a rapid addition in width.

Nanoparticles are used as drug carriers to increase the selectivity and effectiveness of therapy, as well as for combined therapy that utilizes different effects. Iron oxide nanoparticles are promising in this aspect. Due to magnetic properties, they can be used as a contrast agent for magnetic resonance imaging. Also, iron oxide nanoparticles could be coated with a photosensitizer for photodynamic therapy and their laser or magnetic heating can be used for phototherapy. Local enhancement of the electromagnetic field near iron oxide nanoparticles can increase the fluorescence intensity of photosensitizers and the efficiency of singlet oxygen generation. This paper presents the results of a study of iron oxide nanoparticles focused on the photophysical aspects of the formation of “hot spots” under laser irradiation. The photoinduced effects of iron oxide nanoparticles observed in in vitro experiments lead to the rupture of lysosomes. Theoretical modeling showed that the heating of iron oxide nanoparticles with a radius of 35 nm under the action of laser radiation is about 89°C and 19°C for wavelengths of 458 and 561 nm, respectively. Local field enhancement occurs in pairs of nanoparticles of various sizes and strongly depends on the distance between them. The maximum gain is achieved at small distances between nanoparticles. For a dimer of nanoparticles with radii of 10 and 35 nm at a distance of 1 nm, an enhancement factor of two orders of magnitude was obtained. The investigated phenomenon of «hot spots» is in demand for precision therapy, because the photo-induced processes occur at small distances between nanoparticles, in areas of their high accumulation.

Today, photodynamic therapy is one of the most promising minimally invasive methods of treatment of various diseases, including cancer. The main limitation of this method is the insufficient penetration into the tissue of laser radiation used to activate photosensitizer molecules, which makes it difficult to carry out therapy in the treatment of large or deep-seated tumors. In this regard, there is a great interest in the development of new strategies for photodynamic therapy using infrared radiation for excitation, the wavelengths of which fall into the “transparency window” of biological tissues. In this work, it was proposed to use upconversion NaGdF4 :Yb:Er nanoparticles (UCNP), which absorb infrared excitation and serve as a donor that transfers energy to the photosensitizer. Photosens and phthalosens were chosen as the most promising photosensitizers for the study. The aim of this work was to study the energy transfer processes between upconversion nanoparticles doped with rare-earth ions and photosensitizer molecules. in order to excite photosensitizers with IR radiation and carry out photodynamic therapy of deep-seated neoplasms. Using spectroscopic and time-resolved methods, it has been demonstrated that there is an efficient energy transfer between upconversion particles and photosensitizers phthalosens and photosens. The calculated efficiency of energy transfer by the Foerster mechanism was 41% for the UCNP + photosens system and 69% for the UCNP + phthalosens system. It has been experimentally and theoretically proved that there is a binding of photosensitizer molecules with UCNP by means of surfactants, leading to a reduction in the distance between them, due to which effective nonradiative energy transfer is realized. The generation of singlet oxygen by the phthalosens photosensitizer upon excitation by means of energy transfer from UCNP, excited at 980 nm wavelength of, has been demonstrated.

Neurosurgery of intracranial tumors, especially of glial origin, is a non-trivial task due to their infiltrative growth. In recent years, optical methods of intraoperative navigation have been actively used in neurosurgery. However, one of the most widely used approaches based on the selective accumulation of fluorescent contrast medium (5-ALA-induced protoporphyrin IX) by the tumor cannot be applied to a significant number of tumors due to its low accumulation. On the contrary, Raman spectroscopy, which allows analyzing the molecular composition of tissues while preserving all the advantages of the method of fluorescence spectroscopy, does not require the use of an exogenous dye and may become a method of choice when composing a system for intraoperative navigation or optical biopsy. This work presents the first results of using the principal component method to classify Raman spectra of human glioblastoma with intermediate processing of spectra to minimize possible errors from the fluorescence of both endogenous fluorophores and photosensitizers used in fluorescence navigation. As a result, differences were found in the principal component space, corresponding to tissue samples with microcystic components, extensive areas of necrosis, and foci of fresh hemorrhages. It is shown that this approach can serve as the basis for constructing a system for automatic intraoperative tissue classification based on the analysis of Raman spectra.

In this work, the authors evaluate the effectiveness of various adhesive systems (AS) in the temporary teeth restoration with composite materials. For this study, the authors used temporary teeth extracted for orthodontic reasons or lost due to a physiological change. Enamel samples with dentin were made from these teeth. Rhodamine B fluorochrome was added to each of the adhesive systems used. For the greater practical significance of research results, adhesive systems of several generations (IV, V, and VII) were chosen. In each sample, a cavity was prepared, and adhesive preparation was carried out according to the AS manufacturer instructions, followed by restoration with a composite material. Then, longitudinal slits were made so that the areas of enamel and dentin for which an adhesive protocol and restoration were performed could be observed in their entirety. The effectiveness of various generations was determined by the depth of penetration of AS components into the dentinal tubules. Fluorescent confocal microscopy was used to visualize the penetration ability of AS. Based on the results of the study, the authors concluded that IV and V generations of AS are the most effective.

Currently, the most promising method for the study of pharmacokinetics of drugs with fluorescent properties is the spectral-fluorescent method. In this article, we propose an algorithm for expanding the dynamic range of the spectrum analyzer by automatically monitoring the maximum spectral density in the recorded fluorescence spectrum and automatically controlled changes in the accumulation time depending on this value, followed by compensation of the output signal with regard to this change, as well as hardware circuit solutions that allow this algorithm.Testing of LESA-01-"Biospeс" spectrum analyzer, upgraded using the proposed approach, was carried out on photosensitizer dispersions based on tetra-3-phenylthiophthalocyanine hydroxyaluminium of various concentrations (from 0.01 mg/l to 50 mg/l), approximately corresponding to the concentrations realized in the process of studying pharmacokinetics in calibration samples and tissues of experimental animals.The proposed solutions that implement the algorithm for recording fluorescence spectra with automatic change of accumulation time depending on the signal level, ensured a significant expansion of the dynamic range of the spectrum analyzer (up to 3.5 orders of magnitude) and improved accuracy in pharmacokinetic studies.

The paper presents the results of a comparative study of methods of cluster analysis of optical intraoperative spectroscopy data during surgery of glial tumors with varying degree of malignancy. The analysis was carried out both for individual patients and for the entire dataset. The data were obtained using combined optical spectroscopy technique, which allowed simultaneous registration of diffuse reflectance spectra of broadband radiation in the 500–600 nm spectral range (for the analysis of tissue blood supply and the degree of hemoglobin oxygenation), fluorescence spectra of 5‑ALA induced protoporphyrin IX (Pp IX) (for analysis of the malignancy degree) and signal of diffusely reflected laser light used to excite Pp IX fluorescence (to take into account the scattering properties of tissues). To determine the threshold values of these parameters for the tumor, the infltration zone and the normal white matter, we searched for the natural clusters in the available intraoperative optical spectroscopy data and compared them with the results of the pathomorphology. It was shown that, among the considered clustering methods, EM‑algorithm and k‑means methods are optimal for the considered data set and can be used to build a decision support system (DSS) for spectroscopic intraoperative navigation in neurosurgery. Results of clustering relevant to thepathological studies were also obtained using the methods of spectral and agglomerative clustering. These methods can be used to postprocess combined spectroscopy data.

Early diagnosis of caries and tooth enamel microcracks is of great importance for preventing the destruction of healthy tooth enamel. Inorder to detect microcracks in the enamel and pathogenic microflora foci that can cause caries, nanoform of aluminum phthalocyanine (AlPc) can be used as a marker. In a colloidal solution, the nanoparticles do not fluoresce, unlike their molecular form. To convert the particle into its molecular form, it is necessary to have a solvent or specific environment (bacteria, macrophages, etc.). That is why the hydrophobic nanoparticles of aluminum phthalocyanine (nAlPc) can act as markers for detecting hidden pathogenic microflora during fluorescent diagnostics. Further reduction of the diagnosis time and increase the efficiency can be achieved by using biologically compatible surfactants as additional activators of nAlPc.In order to carry out local fluorescence spectroscopy of enamel microcracks and pathogenic microflora foci on the enamel surface, a model compound containing surfactants, auxiliary components and nAlPc colloid at a concentration of 10 mg/l was prepared.Studies on the interaction of the model compound with nAlPc and Protelan MST-35 with tooth enamel ex vivo have shown this surfactant to be a promising auxiliary activator of the nanoparticles, allowing conducting local fluorescence spectroscopy of the tooth enamel surface 3 min after application. In addition, statistical processing of the results showed the effectiveness of using the model compound for local fluorescence spectroscopy of the enamel surface in order to detect the enamel microcracks and the pathogenic microflora accumulation foci that can lead to the development of a cariogenic process.

Laser hyperthermia is one of the promising methods for treatment of oncological diseases. For routine clinical use of hyperthermia, it is necessary to control the uniformity and localization of heat within the tumor. Local heating can be achieved by using special thermal agents, such as nanoparticles doped with rare-earth ions. Measurement of the temperature of the thermal agents will allow timely regulation of the applied laser radiation excitation power and optimization of the hyperthermia process.The paper presents the results of a study on the non-invasive determination of the YPO4 nanoparticles doped with Nd3+ temperature with sensitivity of 0.2% °С-1 in 30-60°С temperature range. The temperature of the nanoparticles was calculated from the Nd3+ luminescence spectra in the 800-1000 nm range under excitation into 4F5/2 energy state by 805 nm laser. A calibration procedure for recalculating the ratio of the luminescence intensities from the Stark sublevels of the 4F3/2 Nd3+ state into the values of the real NP temperature in accordance with the Boltzmann distribution is given. An algorithm for calculating luminescence intensities for individual Stark components is proposed. After calculating the intensities corresponding to each individual Stark component, all the intensities related to the transition from the upper and lower Stark sublevels of the 4F3/2 state are summed, and then their ratio is calculated. The resulting ratio is normalized to the value of the ratio at room temperature and, in accordance with the calibration dependence, is recalculated into the NP heating temperature. It was demonstrated that the investigated 1%Nd3+:YPO4 nanoparticles can be used as "primary” thermometers that do not require additional recalibration to evaluate the temperature in the range used for hyperthermia.

The abilities of the optical probe for fluorescence-guided stereotactic biopsy were investigated by means of a multifiber probe under 532 or 632.8 nm excitation. The set of multilayered phantoms representing a border between normal brain-tissue and a tumor with photosensitizer and gold nanoparticles (spheres or stars) in different concentrations were made to investigate the macroscopic parameters observed during the neurosurgical biopsy sample collection by means of the optical probe. These investigantions will allow to define the border of a brain tumor and control the type of tissue being collected by a stereotactic cannula during surgery. The impact of gold nanospheres and gold nanostars added in different concentrations to the bottom-layer of phantom was analyzed by fluorescence quenching or enhancement due to energy transfer between nanoparticles and fluorescent molecules. The results allow determining the tumor border and to make the biopsy uptake more efficient by observing the character of signal change while penetrating a tumor and while going out of it.

Recent developments in the field of biophotonics facilitate the raise of interest to inorganic nanoparticles (NPs) doped with Nd3+ ions, because of their near-infrared (NIR) absorption. These NPs are interesting bioimaging probes for deep tissue visualization, while they can also act as local thermometers in biological tissues. Despite the good possibilities for visualization of NPs with Nd3+ ions in NIR spectral range, difficulties arise when studying the cellular uptake of these NPs using commercially available fluorescence microscopy systems, since the selection of suitable luminescence detectors is limited. However, Nd3+ ions are able to convert NIR radiation into visible light, showing upconversion properties. In this paper we found optimal parameters to excite upconversion luminescence of Nd3++:LaF NPs in living cells and to compare the distribution of the NPs inside the cell culture of human macrophages THP-1 obtained by two methods. Firstly, by detecting the upconversion luminescence of the NPs inVIS under NIR multiphoton excitation using laser scanning confocal microscopy and secondly, using transmission electron microscopy.

The aim of this study was to analyze the distribution of chlorin type photosensitizers (PS): chlorin e6 (Ce6) and dimethyl ester of chlorin e6 (DME), in human adenocarcinoma HT29 monolayer and multicellular spheroid cell cultures. There is an assumption, that the chemical modification of Ce6 molecules causes a change of intracellular location and the enhanced photosensitizing activity. Indeed, photodynamic therapy on monolayer cell culture with DME showed two times higher photokilling ability comparing with that of non-modified analogue.Ce6 and DME biodistribution processes in tumor tissue were studied on multicellular tumor spheroids model. Total amount of DME in multicellular tumor spheroids exceeded 1,3 times accumulation of Ce6. According to fluorescence microscopy studies, Ce6 and DME distribution patterns in the spheroids bulk were similar. Application of 2D and 3D tumor models for the analysis of photosensitizer distribution may allow predicting the photosensitizer biodistribution features for photodynamic therapy in vivo.

The results of evaluation of the soft tissue perfusion of the foot in patients with diabetes and critical lower limb ischemia (CLI) via fluorescence angiography (FAG) in the near-infrared range are represented in the article. The study included 4 diabetic patients with CLI. All patients underwent lower-limb balloon percutaneous transluminal angioplasty. To evaluate the soft tissue perfusion of the foot via FAG the following parameters have been used: T0m - time to reach maximum for fluorescence intensity after intravenous administration of Indocyanine green; TIm- time of onset of maximum fluorescence intensity after its appearance in the area of interest; Im - the level of the maximum fluorescence intensity. The region of interest was the skin around the wound of the foot. The median FAG parameters in the region of interest on the foot were as follows: prior to surgery T0m = 164 sec (148–181), TIm = 48 sec (38–56); after surgery: T0m = 80 sec (69–92); TIm = 27 sec (20–39); (p

In this review, based on more than 70 articles of Russian and foreign authors, methods of skin engraftment monitoring are discussed. Main processes occurring in skin on cellular and subcellular levels at different stages of engraftment are considered. Optical methods which allow performing non-invasive analysis of blood vessels, collagen concentration and form of cellular respiration (by NADH fluorescence) are described. Comparative analysis of nuclear and optical methods for engraftment monitoring highly developed and widespread nowadays is presented. The advantages of optical methods includes multifunctionality, usability and clarity of results, safety and low cost. In contrast to X-ray CT, MRI and ultrasound, optical methods can be used in monitoring mode. One of the promising directions for improving quality of engraftment due to antibacterial effect, photodynamic therapy, is described in details. The use of crystalline organic nanophotosensitizers (particularly aluminum phthalocyanine) is shown to be the most promising. The main distinctive feature of its application is that nanoparticles injected into wound surface or contact area of tissue graft are not photoactive until the moment the inflammation starts. The development of method for assessing skin condition by spectroscopic properties of tissue components (using fluorescent dyes and photosensitizers in molecular and nanoforms), which allows analyzing physiological state of skin (degree and rate of engraftment or rejection) and controlling certain biochemical and physiological parameters of a tissue graft or an entire area of affected skin is shown to be crucial.

The development and the spectral research of unique coating as crystalline nanoparticles of IR photosensitizers were performed for the creation of hydroxyapatite implants with photobactericidal properties. It has been proved that by the interaction of nanoparticles covering implant with the polar solvent, which simulates the interaction of the implant with the biocomponents in vivo (fast proliferating and with immunocompetent cells), photosensitizers nanoparticles change the spectroscopic properties, becoming fluorescent and phototoxic. Thus, the developed coating based on crystalline photosensitizer nanoparticles with studied specific properties should have antibacterial, anti-inflammatory effect by the photodynamic treatment in the near implant area. This research opens the prospect of the local prevention of inflammatory and autoimmune reactions in the area of implantation. The results of the study suggest a promising this technology in order to create implants with photobactericidal properties.

This work is dedicated to the study of the photoluminescence kinetics of aluminum phthalocyanine nanoparticles in colloidal solutions at different pH and in the interaction with immune cells (macrophages). For measurements we used a registration system based on Hamamatsu streak camera (C10627-13 Hamamatsu Photonics) with picosecond temporal resolution (15 ps), conjugated with the fiberoptic spectrometer and picosecond laser pumping. The changes in fluorescence decay kinetics as additional lifetime components of fluorescence were found during the experiment. The number of components and duration of lifetimes changed while interacting with cells and depends on pH. At pH 2 the presence of two fluorescence lifetimes was recorded: the first one was 5 ns, which corresponded to the molecular form in solution, and 1.5 ns, which corresponded to bound state of phthalocyanine molecules. Due to the absence of other possible objects for bounding in the solution except of the nanoparticles we can suggest with a high degree of accuracy that the bounding occurs with the very these nanoparticles. Analysis of the fluorescence lifetimes of aluminum phthalocyanine nanoparticles in macrophages indicated the presence of two components: 9 ns and 4.5 ns. A model of surface molecules transitions from parallel to perpendicular position, regarding to the plane of the crystal nanoparticle was proposed.

Measuring system based on the binocular microscope for analysis of intracellular accumulation of infrared IR photosenstizers allowing to obtain graphic data about state of analyzed objects, location of fluorescence foci and to obtain details of spectral profile of fluorescence emission centers in IR spectral region was developed. According to image of fluorescence signal distribution the location of photosensitizer accumulation in the cell may be detected accurately and the spectrum of fluorescence signal of near IR-range in the targeted point may be obtained. The developed system is quite comprehensive because there is an opportunity to choose technical parameters, operating modes, measuring methods and analysis. The advantage of the developed microscope-spectrum analyzer is an opportunity to focus emission and create high power intensity on the irradiated area by means of laser source with small-angle beam spreading, all this allow to perform ultra-precise operations with cells. Particularly, tunable size of the diaphragm opening in the far field allows to register fluorescence signal on certain cell organoids. By means of developed system the studies of accumulation of the new bacteriochlorine photosensitizers on HeLa cell line were performed. The system allowed to register accumulation of cancer cells with definite sites of selectively accumulated photosensitizer. The sites of fluorescence were the centers of accumulation of bacteriochlorine photosensitizer, this suggests that studied photosensitizer has a tendency for local accumulation in cellular organoids. The authors suggested that the developed system allowed to perform the effective and rapid screening of new photosensitizers, particularly IR bacteriochlorine photosensitizers.

The results of hardware and tool development in photodynamic therapy and fluorescence diagnostics performed by the Natural Research Center, A.M. Prokhorov General Physics Institute, Russian Academy of Sciences in collaboration with several research and medical institutes are presented. Physical and technical aspects of the problem are mentioned. We describe schemes and the principle of operation of devices which we use with our medical colleagues in clinical and experimental studies. Some results of clinical use of the developed devices and methods are presented.

Treatment outcomes in patients with age-related vascular degeneration due to formation of subretinal neovascular membrane (SNM) of two groups: photodynamic therapy (PDT) with Photosens alone (18 patients) and in combination with anti-VEGF therapy with Lucentis (20 patients). For both groups Photosens was administrated i.v. in single dose of 0.05 mg/kg. The irradiation was performed on the 3rd day (the wave length 675 nm, light dose 120 J/cm2, total light dose did not exceed 500 J/cm2). The number of sessions accounted for 3 to 5 per week according to clinical manifestation of SNM. Patients with multimodality treatment had intravitreal administration of Lucensis in dose 0.05 ml (0.5 mg). The study showed that combination of PDT and anti-VEGF therapy improved vision better and with more stable effect then PDT alone. Thus vision improvement and decrease of SNM activity occurred in 50% of patients with PDT alone and in 60% of patients with multimodality treatment. For 2-year follow-up in the group of PDT alone the vision gradient gradually decreased compared with baseline vision (from 0.11 to 0.06), in the group of multimodality treatment gradual increase of vision gradient was noticed (from 0.03 to 0.155). The superior efficiency of PDT combined with anti-VEGF therapy compared with PDT alone in patients with age-related vascular degeneration was confirmed by study of vision, fundus angiography and average thickness of retina in foveola in both groups.

Emission spectral properties and quantum efficiency of upconversion particles NaYF4, SrF2, LaF3, BaF2 и CaF2, doped with rare earth ions pair Yb3+–Er3+ were studied using continuous wave (CW) and pulsed periodic excitation modes in the near infrared (NIR) spectral range. Analysis of the obtained results showed that the intensity ratio of upconversion luminescence in green and red spectral ranges depends on excitation pulse duration. Thus, by changing the pulse duration the spectral properties of upconversion luminescence can be controlled. Crystals with higher phonon energy are more sensitive to the change of pumping mode. Interpretation of results was performed on the rate equation model basis. Using numerical methods for all energy levels involved in the upconversion process the population and depopulation dynamics were obtained with respect to the duration of the excitation pulses. It was shown that about 30 ms was required for the complete population of 4F9/2 state, from which the luminescence in the red spectral range occurs. When the pulse duration was less than 30 ms, the 4F9/2 population did not reach a steady state and the intensity of the luminescence in the red part of the spectrum was reduced. The theoretical dependence of the upconversion luminescence intensity in the green and red ranges of the excitation pulse duration for NaYF4:Yb0.2–Er0.02 composition was obtained and demonstrates good agreement with the experimental results. [ABSTRACT FROM AUTHOR]