We present here the effective video-fluorescence diagnosis with laser excitation at 635 nm with subsequent TUR of the bladder. After the diagnosis, the main tumor foci were resected first, and then a fluorescence analysis of the tumor borders was performed. Small fluorescing tumor tissues, not seen by cystoscopy in white light, were detected and the diagnostic fluorescence contrast was measured to quantify 5-ALA in tissues. The endoscopic images, registering the tumors, are very important in the development of signal processing techniques in augmented reality mode or mosaic mapping of the obtained series of images.

Currently, there is a significant increase in the incidence of cancer of the central nervous system. Determination of the boundaries of intracerebral and intramedullary tumors is especially difficult. The urgency of the problem of determining the boundaries of astrocytic tumors is due to the peculiarities of their growth along myelinated nerve fibers and vessels, leading to the infiltration of healthy white matter by tumor cells, which affects the high frequency of postoperative relapses. The complexity of surgery for intramedullary tumors of the spinal cord is that the tumor does not always have a clear border and the risk of injury is high due to the smaller size of the operated area compared to the brain. Reliable information regarding the volume of the resected tumor should be obtained by intraoperative imaging. The solution to this problem is implemented mainly in three directions: the use of intraoperative computed tomography, magnetic resonance imaging and ultrasound scanning, and various combinations of these methods. Unfortunately, all these methods of intraoperative diagnostics do not allow real-time examination of tissues in an operating wound and/or do not provide a simultaneous analysis of both structural and metabolic changes. The limitations of intraoperative navigation methods in neurosurgery have led to the relevance of the development of an accurate spectroscopic method for in vivo determination of the content of specific metabolic markers and structural changes accompanying the development of the tumor process in the nervous tissue. Various approaches to intraoperative navigation based on optical spectroscopy are called optical biopsy. In this article, we present the methods and tools developed in recent years for spectroscopic guidance in neurooncology. First of all, this, of course, concerns the analysis of spectral dependences recorded before, during and after tumor removal. We have used such modalities of optical spectroscopy as fluorescence, diffuse reflectance spectroscopy and spontaneous Raman scattering. An equally important issue on the way to increasing the efficiency of tumor resection is the development of new instrumentation; therefore, we have developed a number of new devices, which are a combination of well-known neurosurgical instruments and laser and fiber-optic technologies. Last but not least is the issue of rapid classification of the studied tissues based on the recorded signals, which was solved by us using machine learning methods.

Cancer is the main problem of all developed and many developing countries of the world and the cause of death and disability of population. Today, the actual problem is receipt of reliable information about the boundaries of malignant neoplasms and the detection of pathology in the early stages.

Background: The five-year survival rate for successful surgical treatment of cholangiocellular cancer is only 20-40%, and in the case of an unresectable tumor, the life expectancy does not usually exceed 6 months. Survival decreases with the presence of jaundice, due to the spread of the tumor process along the bile ducts, leading to their obstruction. We report outcomes of patients with nonresectable bile duct carcinoma complicated by obstructive jaundice treated with Photodynamic Therapy (PDT). Methods: Combined diagnosis and treatment included percutaneous cholangiostomy, intraductal video fluorescence diagnostics, photodynamic therapy, and bile duct scenting. All patients were treated at the Sechenov University Oncology Center in Moscow. The results of treatment of 33 patients have been presented. The intraductal diagnosis of malignant bile duct lesions was performed after cholangiostomy using the endovideo-fluorescence module for minimally invasive surgery and endoscopy. With the use of this method, it is the first time in Russia that it has become possible to obtain a videofluorescent image of the tumor and to determine the high level of photosensitizer accumulation in all cholangiocarcinoma patients. The preparations Photolon, Radachlorin, and Photosens were employed as photosensitizers (PS). Intraductal photodynamic therapy was used to achieve the antitumor effect. Laser power density was about 200 mW/cm(2). Results: We present initial results, improved the diagnostic possibilities in this difficult localization of carcinoma, and demonstrated the feasibility of prolongation of life without significant deterioration of its quality. The average survival time in the treatment group is 9.5 months. Conclusion: The treatment of patients with nonresectable cholangiocarcinoma with Photodynamic Therapy should be an available option. In this context, the additional use of intraductal endovideofluorescence diagnostics is a highly specific technique that allows reliable detection of the photosensitizer accumulation predominantly by the tumor tissue and appears promising. As shown by our experience, flourescent localization followed by Photodynamic Therapy, enabled us to improve diagnostic techniques and treat the tumor with improved outcome.

Squamous cell carcinoma is the most common type of oral and oropharyngeal cancers. Incomplete surgical removal of the tumor is often the cause of local recurrence of the disease and appearance of metastases. We have developed the novel endoscope fluorescence video system for visualization of micrometer size objects in real-time mode. The novel system was tested on three-dimensional models of cancer cells. Chlorine e6 (Ce6) was used as a photosensitizer. The accumulation of Ce6 in cancer cells was assessed by its fluorescence excited at 635 nm. It was shown that the 500 µm multicellular tumor spheroids could be easily detected with a good resolution and sensitivity. We suppose that the new endoscope video system could be useful for developing the intraoperative fluorescence navigation method using Ce6-mediated techniques that is able to visualize the small cancer cell clusters.

To improve methods of laser hyperthermia for the treatment of bulk malignant neoplasms, an urgent task is the development of techniques and devices that automatically control heating at a given tissue depth and ensure its uniformity. The article proposes the concept of a system for performing hyperthermia with real-time spectroscopic temperature control and surface cooling, which allows to record spectra of diffusely scattered radiation and fluorescent signal from various depths of biological tissues by the means of the variation of the angle and distance between the fiber source of laser radiation and the receiving fiber. Theoretical and experimental modeling of the spatial distribution of diffusely scattered radiation and temperature inside the tissue with a fiber optic device providing surface cooling of the irradiated tissue, and recording spectral information from a given depth in real time, is presented. Simulation of radiation propagation in biological tissues, depending on the distance between the source and the receiver and the angle of their tilt, was carried out using the Monte Carlo method. Modeling of the temperature distribution inside the tissues was carried out by means of a numerical solution of the heat conduction equation. Experimental modeling was carried out on phantoms of biological tissues simulating their scattering properties as well as accumulation of the investigated nanoparticles doped with Nd3+ ions. It was shown that inorganic nanoparticles doped with rare-earth Nd3+ ions can be used as temperature labels for feedback to the therapeutic laser. According to the results of the theoretical simulation, optimal configurations of the relative arrangement of the fibers were chosen, as well as the optimum surface cooling temperatures for the given power densities. The heating of the phantom of the neoplasm containing the investigated nanoparticles doped with Nd3+ ions by laser radiation with an 805-nm wavelength and power density of 1 W/cm2 up to 42 °C at a depth of 1 cm while maintaining the surface temperature within the limits of the norm was demonstrated.

In this paper we consider a method for researching the dynamics of blood flow in the cerebral cortex, on an optical phantom that reproduces the parameters of real human and mice brain structures, through the use of near and infrared ranges of laser radiation. For the investigation of real tissue we chose a laboratory mouse brain in vivo. An algorithm for non-invasive diagnostics of the degree of oxygenation was identified and optimal parameters of installation components were selected for taking information about hemodynamic indicators. Output was verified by the reference method for assessing oxygenation by degree of absorption of hemoglobin in the visible range, which indicates that data have a high correlation with classical methods. With further development, this algorithm can be used in various areas of research and diagnostics.

The proposed method provides neurosurgical intraoperative navigation using optical spectroscopy and neurophysiological stimulation. The device contains a spectrometer, sources of optical radiation and fiber-optic probe, the working part of which is made in one body with a cannula of neurosurgical aspirator and can be used also as a monopolar stimulant for the system of intraoperative neuromonitoring.

Current paper presents the results of the system development for intracranial implantation aimed on therapy and prevention of brain gliomas relapse. The main property of the system, in prospective, will be to direct the growth of glioma cells localized in the region adjacent to the site of the removed tumor along the fi bers towards the proximal part of the fiber-optic scaffold (neuroport). Such approach will allow carrying out cells diagnostics by the photoluminescence signal and provide subsequent destruction of malignant cells by photodynamic action. Besides, this system could be used for monitoring the processes occurring in the probed area in order to control the possible relapses. The localization of cells along the fi ber structures covered with gelatin compound, which is the source of amino acids during cultivation, was shown during the glioma cells growth dynamics study. Moreover, four different designs of intracranial scaffold models, serving as ports for diagnostic and therapeutic laser radiation delivery, were developed and successfully tested in the framework of the research. The results obtained on the rats brain with induced tumors (glioma C6) after neuroport implantation demonstrate sufficiently intense fluorescence in the tumor bed after intravenous injection of the nonmetallic sulfonated phthalocyanine based photosensitizer, and a pronounced photodynamic effect leading to total destruction of the tumor. In this way, the results of this study open the prospects of creating the neuroport with an internal fi ber structure that focuses the glioma cells growth.

ИНТЕРВЕНЦИОННЫЕ МЕТОДЫ ЛЕЧЕНИЯ, ФЛЮОРЕСЦЕНТНАЯ ДИАГНОСТИКА И ФОТОДИНАМИЧЕСКАЯ ТЕРАПИЯ НЕРЕЗЕКТАБЕЛЬНОЙ ХОЛАНГИОКАРЦИНОМЫ

Aim. To improve the diagnostic and treatment approaches for patients with unresectable liver cholangiocarcinoma. Material and Methods. Diagnosis and treatment of 14 patients are presented. Videofluorescent module was developed for diagnostics. For the first time in Russia we received videofluorescent image of bile ducts tumors and determined the accumulation of photosensitizers. In 12 patients the diagnosis was morphologically confirmed. Patients with unresectable cholangiocellular cancer underwent US- and X-ray-assisted percutaneous drainage of bile ducts. Videofluorescent diagnostics, photodynamic therapy of tumoral stricture and stent deployment were made after release of jaundice. Photodynamic therapy was performed using a fiber-optic system. Controlled balloon catheter on the distal segment was used if it was necessary to dilate the stricture and for uniform irradiation. Nitinol stent was deployed after photodynamic therapy. Photosens, Radachlorin and Photolon photosensitizers were used. Results. We acquired videofluorescence image of the tumor and determined the accumulation of photosensitizer in all patients. Cholangiocellular cancer was morphologically confirmed in 12 patients. The effectiveness of unresectable cholangiocellular cancer management was evaluated by survival. One patient was alive for 21 months, 3 patients – for 11, 13 and 17 months respectively. Five patients are under dynamic observation: two – for 12 and 18 months, three – from 3 to 6 months. Five patients died within 3 ± 1 months after treatment and initially had a lot of distant metastases. Conclusion. Videofluorescent diagnosis of cholangiocellular cancer determines the malignant nature of biliary lesion with high probability. Biliary stenting combined with photodynamic therapy allows us to establish a regular biliary passage and antineoplastic treatment that is associated with better results.

The results of the study of subcellular distribution of molecular meso-tetra(3-pyridyl)bacteriochlorin nanocrystals proposed as therapeutic agents for photodynamic therapy are represented in the article. Investigations and measurement of spectroscopic properties of molecular crystals of near-infrared photosensitizer were conducted using special device complex based on fiber-optic spectrometer. Investigation and analysis of the pattern of subcellular accumulation of meso-tetra(3-pyridyl)bacteriochlorin in molecular (dimethyl sulfoxide (DMSO) as solvent) and nanocrystalline forms on different cell lines: human monocytes (THP-1), human cervical cancer cells (HeLa) and mouse malignant brain tumor cells (glioma C6). The dynamics of subcellylar accumulation of the agent at concentration of 5 and 10 mg/l was assessed with laser microscope-spectrum analyzer and by confocal microscopy. The study showed that in the course of interaction with cell lines molecular nanocrystals of the agent developed ability to fluorescence. Hence, in the cellular environment meso-tetra(3-pyridyl) bacteriochlorin nanoparticles became phototoxic giving opportunities for their use for fluorescence diagnosis and photodynamic therapy. Specific role of meso-tetra(3-pyridyl)bacteriochlorin in the range of photosensitizers is determined by its spectral characteristics, i.e. absorption and fluorescence in near-infrared band, which allows measuring and affecting on deeper layers of biotissue. Thus, the use of meso-tetra(3-pyridyl)bacteriochlorin nanoparticles as nanophotosensitizers may improve the efficacy of diagnosis and treatment of deep-seated tumors.

The results of the pilot study of combined treatment for non-resectable cholangiocarcinoma complicated with obstructive jaundice are represented this paper. Method included percutaneous transhepatic biliary drainage, endoscopic fluorescence diagnosis, photodynamic therapy of tumor stricture, and stenting of bile ducts. Fourteen patients who underwent the treatment in the surgery department clinic of I.M. Sechenov First Moscow State Medical University were enrolled in the study. Fluorescence diagnosis and photodynamic therapy were carried out using photosensitizers photosens (0.5 mg/kg), fotolon (1 mg/kg), and radachlorin (1 mg/kg). The average light dose for one session was 115±5 J/cm2. Fluorescence diagnosis using endoscopic video-fluorescence system for endoscopy and minimally invasive surgery allowed to obtain videoassisted fluorescence image of the tumor and to measure level of photosensitizer fluorescence in tumor in all patients. Malignant tumor was confirmed by morphological study in 12 patients, biopsy of material for morphological study failed in 2 patients with Klatskin tumor. The preliminary results of combined minimally invasive treatment were assessed as promising. The survival time in 4 patients after treatment accounted for 21, 17, 13 and 11 months, respectively. For now 5 patients are under follow-up. Follow-up periods are 13 and 19 months in 2 of them and from 4 to 6 months in 3 of them. Five patients with multiple distant metastases before the treatment died in 3±1 months after therapy. The average lifetime in the treatment group is 9.5 months up to date, however the duration is expected to belonger because 5 of 14 patients are alive.

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.