The new approach to intraoperative navigation during glial brain tumors removal is presented. A combined method is proposed for simultaneous spectroscopic and video fluorescence analysis of the state of tissues in the destruction zone using the applied part performed in the form of a neurosurgical aspirator cannula. In the walls of the applied part there are tubular channels into which lighting and receiving optical fibers are integrated. At the end of the cannula, the channels for optical fibers are arranged so as to perform spectroscopic analysis in contact with the surface of the biological tissue, as well as video fluorescence analysis at the working distance to the surface of the tissue. The joint use of fiber-optic systems for recording the video stream and spectral dependences allows real-time assessment of the degree of pathological tissue changes in the field of view of the video system, which are also located in the aspiration zone, with the simultaneous quantification of diagnostically significant spectroscopic criteria. System testing was carried out on samples of human intracranial tumors obtained during neurosurgical operations. During the removal of a tumor from different sites (tumor center, perifocal area), the degree of in vivo fluorescence signal from the tumor site was determined intraoperatively using a Zeiss Opmi Pentero intraoperative microscope in Blue 400 mode. From the selected area of the tumor, biopsy material was taken (presumably homogeneous in its properties) with subsequent measurement of spectra and combined images using the developed device. A high correlation was shown between the level of the fluorescence signal recorded spectroscopically and the brightness of the fluorescence image in the endoscopic channel of the device. The level of the fluorescent signal showed a high correlation with the degree of malignancy of tissues according to the results of pathomorphological examination.
The limited penetration of laser radiation into biological tissue prevents the widespread distribution of photodiagnostics (PD) and photodynamic therapy (PDT) methods to clinical practice. We have investigated several approaches for PD and PDT of deep-seated tumors: 1. Stereotactic biopsy cannula with a laser spectroscopic control. Special fiber ports for long-term installation in the tumor removal area were developed in order to cause tumor cells to migrate not into the depth of brain but along the fibers with occasional laser irradiation for PD and PDT. The fibers are coated with a special compound containing photosensitizer (PS) and nutrients for cancer cells. 2. Neurosurgical aspirator with the function of video-fluorescence and spectroscopic analysis system. More than 500 patients with various types of brain tumors were operated on using fluorescent navigation based on 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (Pp IX) fluorescence under laser excitation in red spectral range. 3. Diagnostics and navigation of tumors when fluorescence is excited in the red and near infrared ranges. We used indocyanine green (ICG) as near infrared dye to observe blood and lymph vasculature of laboratory animals. This method could be useful while examining tumor bed and adjacent area. 4. Joint action of radiopharmaceuticals and PS based on Cherenkov radiation. Cell death by PDT mechanism via Pp IX excitation by Cherenkov radiation in mitochondria during 18F-fludeoxyglucose decay. This idea achieved good results on rats with C6 glioma. The results of using this approach with chlorin e6 PS in comparable doses are negative. 5. Action through photodynamic inactivation of tumor-associated macrophages and microglia. Idea of minimally invasive method for determining macrophage (microglia) phenotype and their polarization in tumors and their immediate vicinity in situ. This would allow evaluating the effectiveness of the treatment, including PDT. The most promising results were obtained with Pp IX and aluminum phthalocyanine nanoparticles. Studies have been conducted on experimental animals with grafted tumors and, in part, on cancer patients in the clinic.
The development of express method for assessing the state of skin graft by the spectroscopic properties of tissue components involved in the healing of the affected skin or healing of skin grafts was carried out in present work. The proposed method for assessing the state of the skin by the spectroscopic properties of tissue components (using photosensitizers, fluorescent dyes (methylene blue and IcG) and nanophotosensitizers aluminum phthalocyanine nanoparticles (NP-AlPc) applied locally) will evaluate the physiological condition of the skin and assess the degree and rate of engraftment or rejection while also controlling several biochemical and physiological parameters in the entire graft, or the whole area of the skin lesions. Such parameters include the oxygenation of hemoglobin in the tissue microvasculature; the blood supply level; blood flow and lymph flow; assessment of intracellular metabolism; assessment of the cellular respiration type (aerobic/anaerobic).To assess the extent of inflammation the spectrally sensitive to biological environment nanoparticles of aluminum phthalocyanine (NP-AlPc) were also used.
The complexity of the biological tissue spectroscopic analysis due to the overlap of biological molecules’ absorption spectra, multiple scattering effect, as well as measurement geometry in vivo has caused the relevance of this work. In the neurooncology the problem of tumor boundaries delineation is especially acute and requires the development of new methods of intraoperative diagnosis. Methods of optical spectroscopy allow detecting various diagnostically significant parameters non-invasively. 5-ALA induced protoporphyrin IX is frequently used as fluorescent tumor marker in neurooncology. At the same time analysis of the concentration and the oxygenation level of haemoglobin and significant changes of light scattering in tumor tissues have a high diagnostic value. This paper presents an original method for the simultaneous registration of backward diffuse reflectance and fluorescence spectra, which allows defining all the parameters listed above simultaneously. The clinical studies involving 47 patients with intracranial glial tumors of II-IV Grades were carried out in N.N. Burdenko National Medical Research Center of Neurosurgery. To register the spectral dependences the spectroscopic system LESA- 01-BIOSPEC was used with specially developed w-shaped diagnostic fiber optic probe. The original algorithm of combined spectroscopic signal processing was developed. We have created a software and hardware, which allowed (as compared with the methods currently used in neurosurgical practice) to increase the sensitivity of intraoperative demarcation of intracranial tumors from 78% to 96%, specificity of 60% to 82%. The result of analysis of different techniques of automatic classification shows that in our case the most appropriate is the k Nearest Neighbors algorithm with cubic metrics.
The method of intraoperative analysis of tumor markers such as structural changes, concentrations of 5- ALA induced protoporphyrin IX and hemoglobin in the area of tissue resection was developed. A device for performing this method is a neurosurgical aspiration cannulae coupled with the fiber optic probe. The configuration of fibers at the end of cannulae was developed according to the results of numerical modeling of light distribution in biological tissues. The optimal distance between the illuminating and receiving fiber was found for biologically relevant interval of optical properties. On this particular distance the detected diffuse reflectance depends on scattering coefficient almost linearly. Array of optical phantoms containing hemoglobin, protoporphyrin IX and fat emulsion (as scattering media) in various concentrations was prepared to verify the method. The recovery of hemoglobin and protoporphyrin IX concentrations in the scattering media with an error less than 10% has been demonstrated. The fat emulsion concentration estimation accuracy was less than 12%. The first clinical test was carried out during glioblastoma multiforme resection in Burdenko Neurosurgery Institute and confirmed that sensitivity of this method is enough to detect investigated tumor markers in vivo. This method will allow intraoperative analysis of the structural and metabolical tumor markers directly in the zone of destruction of tumor tissue, thereby increasing the degree of radical removal and preservation of healthy tissue.
We developed a system for the analysis of the spatial distribution of photosensitizers (PS) based on tetrapyrrole compounds in the tissues of the retina. Tetrapyrrole compounds were chosen because the most of them are characterized by strong absorption in the tissue transparency window. Calibration curves for determining the concentration of PS by different methods were constructed. Registration system of PS’s fluorescence consists of two optical channels. First channel based on single point spectroscopic technique is used to determine exact concentration of PS at a certain point of the field. Second channel based on imaging sensor is used for concentration mapping in whole field of view. The joint use of these two methods allows determining the concentration of PS in different points of the field quickly and accurately, which provides the exact determination of tissues in pathologic condition and informed choice of irradiation dose for photodynamic therapy.
Brain glial tumors have peculiar features of the perifocal region extension, characterized by its indistinct area, which
complicates determination of the borders for tissue resection. In the present study filter-reduced back-scattered laser light
signals, compared to the data from mathematical modeling, were used for description of the brain white matter. The
simulations of the scattered light distributions were performed in a Monte Carlo program using scattering and absorption
parameters of the different grades of the brain glial tumors. The parameters were obtained by the Mie calculations for
three main types of scatterers: myelinated axon fibers, cell nuclei and mitochondria. It was revealed that diffuse-reflected
light, measured at the perifocal areas of the glial brain tumors, shows a significant difference relative to the signal,
measured at the normal tissue, which signifies the possibility to provide diagnostically useful information on the tissue
state, and to determine the borders of the tumor, thus to reduce the recurrence appearance. Differences in the values of
ratios of diffuse reflectance from active growth parts of tumors and normal white matter can be useful for determination
of the degree of tumor progress during the spectroscopic analysis.
KEYWORDS: Oxygen, Tissues, Diffuse reflectance spectroscopy, Scattering, Imaging systems, Monte Carlo methods, Absorption, Imaging spectroscopy, Real time imaging, Light scattering
The system of multi-spectral imaging of oxygen saturation is an instrument that can record both spectral and spatial information about a sample. In this project, the spectral imaging technique is used for monitoring of oxygen saturation of hemoglobin in human tissues. This system can be used for monitoring spatial distribution of oxygen saturation in photodynamic therapy, surgery or sports medicine. Diffuse reflectance spectroscopy in the visible range is an effective and extensively used technique for the non-invasive study and characterization of various biological tissues. In this article, a short review of modeling techniques being currently in use for diffuse reflection from semi-infinite turbid media is presented. A simple and practical model for use with a real-time imaging system is proposed. This model is based on linear approximation of the dependence of the diffuse reflectance coefficient on relation between absorbance and reduced scattering coefficient. This dependence was obtained with the Monte Carlo simulation of photon propagation in turbid media. Spectra of the oxygenated and deoxygenated forms of hemoglobin differ mostly in the red area (520 - 600 nm) and have several characteristic points there. Thus four band-pass filters were used for multi-spectral imaging. After having measured the reflectance, the data obtained are used for fitting the concentration of oxygenated and free hemoglobin, and hemoglobin oxygen saturation.
Photodynamic therapy (PDT) is one of the advanced methods of treatment of skin cancer and surfaces of internal organs.
The basic advantages of PDT are high efficiency and low cost of treatment. PDT technique is needed for providing
fluorescent diagnostics. Laser-based systems are widely applied to the fluorescence excitations for diagnostic because of
a narrow spectrum of fluorescence excitation and high density of radiation. Application of laser systems for carrying out
fluorescent diagnostics gives the image of a tumor distorted by speckles that does not give an opportunity to obtain full
information about the form of a tumor quickly. Besides, these laser excitation systems have complicated structure and
high cost. As a base for the development and creation of a video fluorescent device one of commercially produced
colposcopes was chosen. It allows to decrease cost of the device, and also has enabled to make modernization for already
used colposcopes. A LED-based light source was offered to be used for fluorescence excitation in this work. The
maximum in a spectrum of radiation of LEDs corresponds to the general spectral maximum of protoporphyrin IX (PPIX)
absorption. Irradiance in the center of a light spot is 31 mW/cm2. The receiving optical system of the fluorescent channel
is adjusted at 635 nm where a general spectral maximum of fluorescence PPIX is located. Also the device contains a
RGB video channel, a white light source and a USB spectrometer
LESA-01-BIOSPEC, for measurement of spectra of
fluorescence and diffusion reflections in treatment area. The software is developed for maintenance of the device. Some
studies on laboratory animals were made. As a result, areas with the increased concentration of a PPIX were correctly
detected. At present, the device is used for diagnostics of cancer of female reproductive system in Research Centre for
Obstetrics, Gynecology and Perinatology of the Russian Academy of Medical Sciences (Moscow, Russia).
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