Stem cell and stem cell derived therapies have shown great promise in lab and pre-clinical studies for many diseases, but few are translated to the clinic. The regulatory hurdles for cell therapies are substantial and include the need to monitor biodistribution and efficacy in humans. However, tracking of cells at depths more than 1 mm is difficult and there is a great need for technologies to track cells at clinically relevant depths. Gold nanorods typically give optoacoustic signals of an order of magnitude better than dyes. Recently, we have developed gold nanostars which behave as three gold nanorods stuck together and the energy deposition is better than all other particles of which we are aware. Hence they generate optoacoustic signals which are stronger and we have been able to tune them to absorb energy at 1064 nm to maximize penetration depth and the available laser. This paper will describe the progress to date on tuning, internalization and imaging of cells labelled with gold nanostars.

One of the most common reasons of congenital birth defects is prenatal substance abuse. The severity of the defect depends on the amount of substance abused and the period of gestation during which substance is abused. Although prenatal substance abuse is common during the first trimester, some women continue the abuse well into their second trimester, which is considered the peak period for fetal neurogenesis and angiogenesis. Thus, evaluating the changes in fetal brain vasculature caused by maternal exposure to different teratogens at the second trimester equivalent period is crucial. In this study we use correlation mapping optical coherence angiography (cm-OCA), a functional extension of optical coherence tomography, to image changes in murine fetal brain vasculature caused due to prenatal exposure to ethanol, cannabinoids, or nicotine. Results showed significant vasoconstriction in all three cases.

In this study, we utilize the properties of polarized light for the analysis of paraffin-embedded breast cancer samples. We perform the measurements of the full Stokes vector of back-reflected radiation and calculate the degree of polarization as a diagnostic criterion for the separation of healthy and cancer sample sections. We show that circularly polarized light scattered within the breast sample is sensitive to the presence of cancer cells. The degree of the polarization of the reflected light was found to be the most sensitive parameter for the reliable differentiation of tissue. We show that circularly polarized light scattered within the breast sample is sensitive to the presence of cancer cells. The highest contrast between cancerous and normal regions was observed for the probing wavelength of 450 nm. The degree of the polarization of the reflected light was found to be the most sensitive parameter. Mapping the Stokes vectors of backscattered light on a Poincaré sphere helps to highlight the changes of polarization state.

In this work, we propose using spectro-polarimetric imaging to monitor tumors growth of non-pigmented intra-dermal grafted tumors on murine models. We use for this purpose a full Mueller imaging polarimeter operating in the near infrared range. The injection site was imaged twice a week with our system from day 1 to the sacrifice of the mice while, in parallel, tumor volume was evaluated using standard caliper measurements. 40 nude mice were injected with a nonpigmented cancerous cell line (MDA) to produce intra-dermal tumors and separated into three different groups. The first group, called “early”, received a Docetaxel treatment as soon as the polarization signal detected a change in the tissue matrix, the second one received the Docetaxel treatment as soon as the tumor reached an estimated volume of 20 mm³ and the last group, was used as a control group. We demonstrate that early detection of tumor development based on depolarization metrics permits to control the tumor growth. We show also that while the tumor grows the depolarization contrast decreases while spectral contrasts appear. We observe also a change in fiber orientation around the tumor at the necrotic stage. This is highlighted by the azimuthal retardance orientation extracted from the polar decomposition of the Mueller matrix. This study confirms the usefulness of polarimetric contrast for tumor detection and monitoring. In the future, a precise characterization of different stages of tumor development could be useful for drug efficiency assessment in small-animal studies or in clinical applications.

This work contains the main results of the design of the mobile system for early diagnosis of pigmented skin lesions. The system includes the following main stages: registration of the images of a lesion and the standard placed on the skin surface, evaluation of the image quality, detection of a pigmented lesion, determination of lesion’s parameters, and calculation of the malignancy probability. The advantages of the proposed system for early diagnosis of pigmented skin lesions are noninvasiveness and the ability to automatically determine the parameters of lesions and detect precancerous changes on their basis. The mobile system was tested on 520 digital images of in vivo skin pigmented lesions, including ordinary dysplastic nevi and melanoma. The sensitivity and specificity of the method proved to be 95 and 91%, respectively.

Radiotherapy is already well-established and an effective form of treatment for many cancers, especially for brain tumors. Currently, the clinical efficacy of a treatment, however, can only be established based on clinical or radiological responses observed after a significant period of time following the single irradiations during radiotherapy course. On the other hand, the radiotherapy dose is limited by its toxicity to surrounding healthy tissues. Particularly, radiation to brain tumors may sub-acutely or chronically affect cognition and cause fatigue even with conventional doses. However, there is currently no on-line and safe method to monitor the effects of radiation to the brain during the irradiation. In our project, we aim to develop an on-line method to monitor effects in brain tissue that correlate with the radiation dose in radiotherapy. In this case study, we use functional near-infrared spectroscopy (fNIRS) and study possible temporal effects in cerebral haemodynamics during irradiations of whole-brain radiotherapy (WBRT). fNIRS is safe for the patient, it can be used noninvasively and also in demanding environments, such as in radiotherapy treatment rooms during irradiation, and thus could be in future potential technique to be utilized for monitoring tailored radiotherapy.

Photoacoustic imaging is a new, rising imaging technique which combines high penetration depth with a good image contrast. It is demonstrated for the first time that remote photoacoustic sensing by speckle-analysis can be performed in the MHz-sampling range using a low-resolution diode array by showing experimental results. Phantoms and ex-vivo samples are measured in transmission-mode. In addition, the detection sensitivity of the new system is estimated. The new technique might help in future to broaden the applications of photoacoustics in special applications like wound imaging, endoscopy or guiding laser surgeries.

Toward improved management of developmental defects and reproductive disorders, we recently established a set of unique in vivo methods for investigation of early embryonic development and reproduction in mouse models. Using functional optical coherence tomography (OCT), we investigate female mouse reproductive tract in vivo through an implantable intravital window. Our approach allows for live, dynamic volumetric imaging of the mouse Fallopian tube (oviduct) with micro-scale spatial resolution. Recently, we established a tomographic imaging technique capable of mapping both the cilia location and the cilia beat frequency in the intact mouse oviduct in vivo. We utilize both the amplitude and the frequency position of the major peak from the ciliary motion spectrum, and reconstruct, for the first time, high-resolution mapping of both cilia location and CBF through tissue layers in vivo. We established a method of sperm activity measurement, which relies on analysis of unique tortious sperm trajectories. We introduced standard deviation (SD) of the direction variation (SDofDV) as a measure of activity. This method was used in vivo to directly track multiple motile sperm inside the mouse oviduct, revealing new biological findings. Potentially this study will provide new insight on the process of mammalian fertilization in its native state and lead to a better understanding of pathologies resulting in infertility.

Gemination tests and vigor tests are destructive sampling tests used to ensure the quality of seeds. However, the yields may depend on the conditions of the fields even the quality of seeds were tested. A non-destructive method to inspect the properties of seeds in the fields can improve the yields of crops. A biospeckle OCT was developed to measure the biological activities of germinating seeds. Twenty-nine radish seeds were measured by the OCT under 3 conditions. Nine seeds were treated with hot water to terminate the biological activities before the measurement of OCT and imbibed in room temperatures. Ten seeds were germinated for each condition at room and low temperatures. Low temperatures were to slow down the biological activities. The speckle contrast, defined as the ratio of the standard deviation to the mean of OCT intensities, was used to quantify the biological activities. The modal values of speckle contrast largely increased in 1.8 days when the seeds were germinated at room temperatures. When the seeds were germinated at low temperatures the modal values of speckle contrast took 14 days to increase to the same level of that of the seeds germinated at room temperatures. The modal values of speckle contrast remained low for 14 days of OCT measurements when the seeds were heated. As a conclusion, the results showed that the speckle contrast of OCT intensities quantified the biological activities of seeds during the process of germination and it may be used to evaluate the ability of seed germination.

Histological analysis with white light microscopy of few micron-thick sections of tissue remains a gold standard for tissue biopsy. However, this approach requires a cumbersome multi-step preparation of the sample and relies heavily on the skills of a pathologist. In general, any tissue pathology (e.g. inflammation, fibrosis, cancer) affects the constituents of tissue and changes both optical properties of cells and extracellular matrix. We suggest using the images of transmittance, retardance and depolarization of unstained tissue samples, acquired with the Mueller microscopy, as an input data for automated diagnostic image segmentation. The potential of combining polarimetric data with non-linear microscopy data, such as Second Harmonic Generation (SHG) and Two Photon Emission Fluorescence (TPEF), is also explored for the diagnostic of tissue specific components (e. g. collagen, elastin). The unstained histological cuts of full thickness skin equivalents and cervical tissue of mouse were used for multi-modal microscopy imaging. First, the statistical approach was tested for a segmentation of the images of artificial skin using the images of retardance, depolarization, and transmitted unpolarized intensity. The algorithm of density-based spatial clustering of applications with noise (DBSCAN) clearly discriminates the regions of dermal and epidermal layers of skin cuts. The same approach was tested on the extended set of data including polarimetric, SHG and TPEF images of the histological cuts of mouse uterine cervix. The typical circumferential arrangement of collagen fibers around the cervical os as well as elastin lining of the internal cavities was observed on the segmented images. Merging polarimetric data with SHG data makes clear the appearance of fine structures in the cervical os region. The work is ongoing on testing different data post-processing algorithms for increasing both contrast and accuracy of tissue image segmentation, thus, paving the way for the digital histology for tissue diagnostic.

Agar-based skin phantoms with different thicknesses and hemoglobin concentration were evaluated for diagnostics of skin lesions by RGB imaging. Scattering properties of the phantoms were simulated using intralipid, absorption properties – using lyophilized powder of human hemoglobin. RGB images of phantoms were captured by self-developed laboratory made devices. The algorithm for calculation of chromophore concentrations are based on Beer-Lambert law and includes the photon path length evaluated from the measured photon-time-of-flight signals. Optical properties and chromophore concentration maps of phantoms obtained from RGB images were analyzed. The influence of chromophore concentration on scattering and absorption, photon path length and chromophore maps are discussed.

The interest of using light in clinical practice is increasing strongly and many applications work at various wavelengths from the ultraviolet to the infrared. Due to this great range of applications, the determination of the optical properties of biological tissues in a wide spectral range becomes of interest. The liver is an important organ, since it has a major role in the human body and various pathologies are known to develop within it. For these reasons, this study concerns the estimation of the optical properties of human normal and pathological (metastatic carcinoma) liver tissues between 200 and 1000 nm. The obtained optical properties present the expected wavelength dependencies for both tissues – the refractive index, the absorption and the scattering coefficients decrease with the wavelength and the anisotropy and light penetration depth increase with the wavelength. Although similar behavior was observed for the various properties between the normal and pathological tissues, evidence of smaller blood content in the pathological tissues was found. A possible explanation is that the cancer cells destroy liver’s vasculature and internal architecture, providing though a reduction in the blood content. For low wavelengths, it was observed a matching between the scattering and the reduced scattering coefficients, which implies a nearly zero anisotropy in that range. The scattering coefficient decreases from nearly 140 cm^-1 (at 200 nm) to 80 cm^-1 (at 1000 nm) for the normal liver and from nearly 140 cm^-1 (at 200 nm) to 95 cm^-1 (at 1000 nm) for the pathological tissue.

We developed stain-free optical biomarkers for high-grade T1 bladder cancer tissues based on interferometric phase microscopy (IPM). IPM records a topographic map that represents, for each spatial point, the integral of the refractive-index values along the tissue sample thickness, thus accounting for both the tissue internal morphology and contents. Following the extraction of various parameters from this IPM-based topographic maps of healthy and bladder cancer tissues, we employed a machine learning multivariant analysis for stain-free grading of the bladder cancer invasiveness. Our tools are expected to be used for rapid and inexpensive diagnosis of bladder cancer tissues.

The rapid technical development of human society on Earth leads to the pollution of its atmosphere and an increase in the optical radiation of the Sun and its main part — light and ultraviolet radiation. In order to properly protect and control the effects of optical radiation on the human body, it is necessary to know and understand the process of absorption and conversion of optical radiation falling on the surface of the body. The material contains the original results of experimental studies optical radiation transmission through a sample of quasi-vital skin with pigs of different ages. The reliable results of the percentage ratio of the amount of optical radiation of the optical spectrum that passes under the skin through the skin layer and the individual one wool depending on the species and age of the animal are obtained. The results of the experiment showed that the optical radiation of the Sun affects the body of the animal through the skin, as well as inside the cylinders of a separate wool. This new knowledge is important to monitor and control optical radiation for young and old animals with different wools. A new approach has also been proposed for the dosing method of ultraviolet radiation in accordance with the body's needs for vitamin D.

The ability of diagnostics of melanoma and nevus based on spectral analysis of paraffin-embedded tissues in the 0.3-1.5 terahertz (THz) range has been carried out. The principal component analysis was applied to reduce the dimension of the feature space. A comparison of these spectra shows evident differences between samples. The possibility of applying the optical clearing to paraffin-embedded tissue for improving the visualization of the internal structure of tissue for diagnostic and research purposes is shown. In the studies, a sufficiently strong effect of optical clearing of paraffin-embedded muscle was obtained (63%).

Conventional techniques are insufficient to precisely describe the internal structure, heterogeneous cell populations and the dynamics of biological processes of the diseased liver during and after surgery. There is a need for a rapid and safe method for the successful diagnosis of liver disease to plan surgery, efficacy of regeneration and avoid postoperative liver failure. Here, we analyze acute and chronic liver pathology (cholestasis, cirrhosis, fatty liver disease) during progression by multiphoton microscopy with FLIM and SHG modes and chemical analysis by TOF-SIMS to obtain new data about hepatocyte pathological changes at the cellular and molecular level. All of these techniques enable to study cellular metabolism, lipid composition and collagen structure without sample staining and incorporation of fluorescent or another markers that may allow to use these methods in clinic. Complex of multiphoton microscopy and mass spectrometry provides complete information about the liver structure and function that could not be assessed using each method individually. The data can be used for both to obtain new criteria for the identification of hepatic pathology and to develop a rapid technique of liver quality analysis to plan surgery and avoid postoperative liver failure in clinic.

Terahertz (THz) biophotonics attracts considerable interest as a promising tool for the diagnosis of malignancies with different nosology and localization. Nevertheless, the majority of modern THz spectroscopy and imaging modalities rely on lens- and mirror-based optical systems, which obey the Abbe diffraction limit and provide the spatial resolution of >λ; λ is electromagnetic wavelength. The resolution of several hundreds of microns, or even of several millimeters, strongly limits capabilities of THz technology in malignancy diagnosis, pushing further developments into the realm of sub-wavelength-resolution THz imaging. In our work, we developed a method of THz solid immersion microscopy for continuous-wave reflection-mode imaging of soft biological tissues with a sub-wavelength spatial resolution. In order to achieve strong reduction in the dimensions of beam caustic, an electromagnetic wave is focused into the evanescent field volume behind a medium with a high refractive index. We have experimentally demonstrated a 0.15λ-resolution of the proposed imaging modality at λ = 500 μm. The proposed technique does not involve any sub-wavelength near-field probes and diaphragms, thus, providing high energy throughout. We have applied the developed method for the THz imaging of various soft biological tissues: a plant leaf blade, cell spheroids, tissues of the breast ex vivo, human brain gliomas ex vivo and glioma models from rats ex vivo. The observed results justify the capabilities of the proposed THz imaging modality in biology and medicine.

Quantifying the biomechanical properties of the cornea is required for accurate assessment of tissue health. In this work, we evaluate cornea biomechanical properties during intraocular pressure fluctuation. This method results in a more physiologically relevant assessment of tissue mechanics, and thus diverges from typical elastography techniques that use externally induced deformation. Optical coherence elastography is used to detect displacements in the cornea as IOP is cycled during the heartbeat. The axial strains within the cornea are calculated from these displacements, so that dynamic changes in stiffness can be determined. The results indicate a gradient in axial strain within the cornea.

The use of optical clearing agents makes it possible to increase the probing depth of non-invasive optical diagnostic methods. However, the protective epidermal barrier of the skin prevents the penetration of hydrophilic immersion liquids into the deeper layers of the skin. The aim of this study is to improve the efficiency of skin optical clearing by increasing the permeability of the epidermis for polyethylene glycol with MW 300 (PEG-300) using different physical approaches. We present the results of a comparative analysis of optical immersion clearing of skin in laboratory rats in vivo with and without preliminary ablation of epidermis, fraction microablation and sonophoresis. Laser ablation and microablation has been implemented using a setup based on a pulsed erbium laser (λ= 2940 nm) with different hand-pieces. Sonophoresis has been applied during 2 min with the following parameters: 1 MHz, 2 W/cm2, continuous mode. As an optical clearing agent (OCA) polyethylene glycol (PEG-300) has been used. Basing on optical coherence tomography, we have estimated the attenuation coefficient in the process of optical clearing in two regions at depths of 50–170 µm and 150–400 µm. The results have shown that both surface ablation of skin in vivo lead to the local edema of the affected region that increases the scattering coefficient. However, the intense evaporation of water from the ablation zone facilitates the optical clearing at the expense of tissue dehydration, particularly in the upper layers. Besides, fractional ablation enhances the both rate and degree of optical clearing in upper dermis layers but not affects the deeper dermis. Sonophoresis provides the most effective penetration of OCA into deeper dermis. Time dependences of attenuation coefficient in two regions of dermis at the separate and combined laser and ultrasound impact on the epidermal permeability for PEG-300 have been obtained.

“FDISCO: advanced solvent-based clearing method for imaging whole organs” was recorded for SPIE Photonics Europe Digital Forum, held 6-10 April 2020.

We demonstrate a method to determine the angular- and spectral-resolved scattering properties in the second optical window (1100 nm to 1350 nm) which enabled the characterization of biological nano- and microscaled cell structures. The nanosecond pulses of a spectrally filtered IR enhanced supercontinuum (SC) light source were captured time-resolved to depress background and minimize disruptive effects of the biological cells. The scattering characteristics of biological nano- and micro-scaled cell structures were recorded spectrally- and angle-resolved, the scattered portion of the light after the sample was recorded in a time-resolved manner at defined angles and wavelength. A spectrally filtered and collimated SC light source was used. The scattering results of cellular structures at defined wavelengths were compared to calculations treating the structures as ideal spherical particles. The scattering characteristics of a monolayer of human chondrocytes and mouse fibroblast cells were measured in a standard cell culture plate. Because of the size and distribution of the scattering structures, Mi scattering was assumed and analyzed using a Fourier transform-based approach. The final result was the development of a contamination-free method to study pathological changes in cell cultures, apoptosis or necrosis. In order to record the state of the cells without losing experimental information, the Principal Component Analysis (PCA) method was applied in addition to Fourier analysis, which allows the dimensionality of complex data to be reduced. The method was investigated to evaluate it for an automated acquisition of cell states. The system was tested to detect structural changes of human chondrocytes and L292 mouse fibroblasts before and after poisoning with staurosporin and Triton X100.

The goal of this study was in investigating potential correlation of the effects induced by optical clearing (OC) of the skin and the underlying peripheral tissues with the changes in T2-weighted (T2w) magnetic resonance (MR) signal measured over the matched area in vivo. OC/MRI experiments were performed in athymic nu/nu mice carrying subcutaneous HEp2 tumor xenografts expressing Tag-RFP marker protein at 2-3 weeks after tumor inoculation. Initially, to investigate the effect of OC induced by a mixture of 70% glycerol, 5% DMSO, 25% water, we performed measurements of Tag-RFP fluorescence intensity (FI) and lifetime (FL) before and after OC using a macroscopic confocal scanning system equipped with a supercontinuum laser with the acousto-optic tunable filter and a photon-counting detector. The OC effect was achieved by applying the OC mixture onto the skin over the tumor area for 10 min followed by mixture removal from the skin. Subsequently we performed MRI at 1T using T2w fast spin-echo (FSE) MR pulse sequences before and after OC mixture application in the same animals on two non-consecutive days. Time-correlated single photon counting experiments showed that after OC application FL of Tag-RFP was higher with median difference of 51 ps (P<0.05, Wilcoxon matched-pairs test). Average FI increased by 33% after OC resulting in the higher frequency of fluorescence intensity increase observations (n=19 vs. n=3 with FI decreasing) measured over multiple ROI in 3 animals. The analysis of obtained T2w FSE MR images showed significant quantitative differences (p=0.03) between Gaussian noise-normalized MR signal intensities of the 0.7mm-thick axial peripheral tissue/skin slices before and after OC mixture applications in 2 animals, though in one animal those differences were statistically insignificant. The comparison of T2w MR signal intensities measured in OC mixture phantoms prepared at various dilutions and pure water showed that at chosen FSE MRI parameters the observed differences in MR signal intensity were not due to the application of OC mixture alone and must have been a consequence of OC mixture interaction with the skin and peripheral tumor tissue components. The obtained results point to the potential mechanism of OC clearing as it relates to: 1) a transient change of the peripheral tumoral microenvironment affecting the layer of Tag-RFP expressing cells and resulting in FL increase and T2w MR hypointensity increase caused by shortening of mean proton relaxation times within the voxels of subcutaneous tumors; 2) potential microviscosity increase due to skin permeability for the OC components resulting in the shortening of tissue water proton transverse relaxation times. The phantom experiments suggest that the effect of the OC mixture on MR signal is indirect rather than direct consequence of either OC/water magnetic relaxation properties, or additional chemical shift artifact. Therefore, T2w 1T MRI showed promise as a technique suitable for detecting small longitudinal changes of the MR signal in the subcutaneous tissue under the conditions of OC, which resulted in an increase of FI/FL of a red fluorescent marker protein. The latter effects are expected to benefit in vivo imaging of marker protein expression in animal tumor models.

Enhancing brain fluid movement across blood brain barrier (BBB) has been recognized as a potential treatment of neurodegenerative diseases. Moreover, BBB opening is of high interest also in brain drug delivery in the treatment of brain tumors/cancers. However, efficient therapies which are based on BBB opening are still limited because of insufficient understanding of mechanisms and safety issues. Currently, there are few promising methodologically diverse BBB opening approaches. In this paper, we use functional near-infrared spectroscopy (fNIRS) for the first time for monitoring cerebral hemoglobin and water concentration changes during BBB opening in mouse brain by using two different techniques: intra-arterial mannitol infusion (IAM) and focused ultrasound (FUS). Both of these BBB opening techniques are already in clinical use but their hemo- and hydrodynamic implications have not been investigated from comparative aspect. Two fibre detectors were attached on both sides of the mouse brain and the source fibre was attached on middle of forehead. Further, by using a combination of three wavelengths 690nm, 830nm and 980nm, that have sufficient light penetration in the mouse brain, we can show average dynamics of hemoglobin and water in the whole brain, synchronized with BBB opening. To validate the level of BBB opening we used Evans blue dye and show its accumulation in the brain parenchyma tissue with the corresponding fNIRS responses.

Combined spectroscopic and biochemical measurements were used to improve diagnostic accuracy and to evaluate gastrointestinal tract (GIT) neoplasia development parameters noninvasively. Experiments were performed in mongrel male rats divided into 2 groups – control and experimental. To induce gastric cancer, the rats underwent to chronic stress (overpopulation during 9 months) and diet including the daily using of mtoluidine (25 μg/kg weight) in food and water with a solution of sodium nitrite (0.2%) for 9 months. We studied effectiveness of 5-ALA/PpIX fluorescent analysis of gastric carcinoma and biochemical stress-corresponding indices detection for early diagnosis of primary gastric tumours and their metastatic spreading in liver. Affected by precancerous and cancerous alterations mucosa reveal red fluorescence, related to the accumulation of 5- ALA/PpIX. Liver tissues investigated also presented increase of the red fluorescence, which was used as an indicator for possible pathologic process detection there. The histological examination revealed liver metastases in 67.8 % of the rats with gastric cancer. Biochemical indicators detected malignant alterations presence in GIT, and fluorescent observation addressed the exact area and borders of neoplastic lesions. 5-ALA/PpIX fluorescence detection allow to find and precisely map premalignant and malignant areas of gastric mucosa and liver metastases of stress-induced gastric heterogeneous adenocarcinoma and biochemical evaluation of stressrelated compounds increased the efficiency of such diagnosis and reveal information about the dynamics of lesions development. Diagnostic accuracy achieved using fluorescent detection reaches 93% for gastric carcinoma, and 87% for pre-cancerous mucosa alterations observed.

The paper describes the results of experimental studies using custom developed optical biopsy system for diagnostics in vivo during the procedure of fine needle aspiration biopsy. Experimental studies were conducted in laboratory mice with inoculated hepatocellular carcinoma. The measurements were carried out using fluorescence spectroscopy and diffuse reflectance spectroscopy methods to reveal metabolic and morphological changes in tissues. The results show that the developed approach is sensitive for cancer detection. Quantified differences in the maximum of fluorescence spectra and diffuse reflectance spectra between tumor and normal tissues were demonstrated and approved with morphological analysis.

Articular cartilage is a connective tissue that enables smooth movements between bones in articulating joints. Cartilage consists of extracellular matrix (ECM) and chondrocytes – the cells responsible for synthesis of the ECM. The ECM consists of type II collagen, proteoglycans, water, and some other minor components. Cartilage is prone to degenerative joint conditions, such as osteoarthritis, due to its weak repair capacity resulting from a lack of vascular, neural, and lymphatic networks. Osteoarthritis causes erosion of the cartilage matrix and therefore inhibits its function, resulting in joint pain, loss of mobility, and significant global socioeconomic burden. Currently, surgical treatment of cartilage pathologies is carried out during arthroscopy with variable outcomes. This variability occurs due to the subjective nature of arthroscopy, which relies on manual palpation and visual evaluation of the tissue surface. Diffuse optical spectroscopy in the near-infrared spectral region probes tissue structure and composition via a relationship with its optical properties (the absorption and reduced scattering coefficients). Due to its avascular nature, healthy cartilage is translucent. It thus has low absorption in the near-infrared region, providing the necessary conditions for light to traverse deep into the tissue. This research reports, for the first time, cartilage absorption and reduced scattering coefficients in the near-infrared spectral range and assess their capacity for characterizing the depth-wise profile of cartilage proteoglycan content. The results revealed that cartilage optical properties are strong predictors of its proteoglycan content. The best performance was observed with the prediction of the proteoglycan content by the absorption coefficient.

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.

Iron is highly regulated in the body, since it is an essential element required for life. Fundamental understanding of the key processes that underlie the intracellular transport of iron will have a decisive impact on advancing treatment of diseases that are caused by iron deficiency and iron overload, e.g., anemias and hereditary hemochromatosis. Improved knowledge of iron intracellular transport will also provide insight into many other diseases where iron modulates the pathogenic process, e.g., metabolic syndrome, diabetes, neurodegenerative diseases, and cancer. Measuring the iron-bound form of transferrin in intact biological samples remains a technical challenge that needs to be overcome to understand regulation of endosomal iron release in cells and tissues. Serum transferrin (Tf) is a key regulator of systemic and cellular iron transport. Tf binds ferric iron (Fe3+) for transport throughout the body and delivery into cells via the transferrin receptor (TfR). The iron-bound Tf-TfR complex is endocytosed, and upon acidification of early endosome, the iron is released. Importantly, disruption of iron homeostasis has been linked to cancer progression. Although iron transport has been studied in detail, measurements of iron-bound Tf in tumor tissues are still lacking. Previously, we have developed and validated a Raman hyperspectral imaging technique that identified the iron-bound Tf peak at ~1300 cm^-1 Raman shift. Here, we further investigate the variation in peak intensity within frozen tissue sections of T47D and MDA-MB-231 breast cancer tumor xenografts, which represent luminal and basal cancers, respectively. Our results indicate that Raman spectral imaging can be used to evaluate the iron-bound form of Tf in xenograft sections. Measurements of iron-bound Tf in tumor tissues will permit further characterization of iron transport in breast cancer.

We investigated melanin-pigmented skin samples ex vivo, of benign and dysplastic nevi, as well as malignant melanoma, obtained after surgical excision, containing so called safety areas, where a normal skin could be observed, to obtain a complex and complete view about the feasibility of different excitation sources solely and/or in combination to induce fluorescence signal useful for diagnosis of pigmented cutaneous neoplasia. Using the specialized multispectral analysis of the data obtained by fluorescence using excitation in broad spectral range, covering ultraviolet, visible and near-infrared spectral range, contribute considerably to the both the fundamental determination of tumour tissues’ basic spectral parameters, and to increasing the accuracy and specificity in determining the type of pathology when spectral techniques are used that are applicable to the clinical practice. Excitation wavelengths applied namely were 365, 385, 405, 630, 785 nm. As excitation sources were used set of laser and light emitting diodes with output power in the frames of 20-50mW. Microspectrometers USB4000 and QE65000 (Ocean Optics Inc, USA) in the range of 350-1100 nm were used as detectors of the fluorescence spectra obtained using UV-VIS-NIR excitation. These are preliminary investigations based on ex vivo samples, which would be followed by in vivo investigations and comparative studies to obtain the diagnostic accuracy, using optical spectral techniques, which would surpass the existing clinical techniques for early detection and evaluation of skin cancer. The achieved results demonstrate autofluorescence spectra efficiency in highlighting biochemical changes during tumor growth in a broad spectral range.

A comparative in vivo study of skin tumors and normal skin was performed using Raman and autofluorescence spectral signals simultaneously recorded in the range of 800–1000 nm with a help of portable spectrometer and 785 nm excitation laser. The 1012 spectra were collected from 506 patients of the Samara Clinical Oncological Dispensary. The classification of spectral data for various tumors and normal skin was carried out by the PLS-DA method. The bands with most important spectral differences between analyzed skin tissues were found with the projection on latent structures analysis. The spectral differences showed significant changes among normal skin and tumors associated with such biochemicals as elastin, collagen, melanin, nucleic acid, lipids, phenylalanine. Results of the analysis were presented by utilizing sensitivity, specificity and accuracy and receiving operating characteristic (ROC) curve. To classify 314 spectra of benign tumors and 314 spectra of normal skin the mean accuracy was found to be a 0.86; for classification of 192 spectra of skin cancer and 192 spectra of normal skin the mean accuracy was found to be a 0.84; when the benign tumors and skin cancer were combined and differentiated from all normal skin 0.79 mean accuracy was obtained. The achieved results demonstrates Raman and autofluorescence spectra efficiency in highlighting biochemical changes during tumor growth.

Emerging biomedical instrumentation for imaging and diagnostics requires tissue-mimicking phantoms with optical properties close to those of real biological tissues. Many such tissues and organs consist of several layers with different physical properties, e.g. the head includes the brain, skull, and skin, which in turn also comprise several layers. Structure, optical and mechanical properties depend on the purpose of phantoms and studied materials and technologies. In this paper, we present both plain single-, multi-layered phantoms, and complex opto-mechanical phantoms that imitate parts of the body, in particular, the head. We describe the procedure, materials, internal structure, and tuning of the optical properties of two different phantom manufacturing methods as well as show examples of tissue-mimicking phantoms for different biomedical applications.

The vascular function after interventions as revascularization surgeries is checked intraoperatively and qualitatively by observing the blood ow dynamic in the vessel via Indocyanine Green (ICG) Fluorescence Angiography. This state-of-the-art technique does not provide the surgeon with objective information whether the revascularization is sufficient and should be improved by obtaining a quantitative intraoperative optical blood ow measurement. Previous approaches using ICG Fluorescence Angiography show that the blood flow measurement does not match the reference and overestimates the ow. The experiments indicate that the amount of overestimation is linked to the vessels diameter. We have, in previous work, quantified the propagated error on the flow calculation resulting from the error in the measurement of the vessels diameter and length and realized that they cannot be accounted solely for this deviation. The influence of the transit time error is not revealed yet. We propose a model combining the penetration depth of diffusely reflected photons and the flow velocity profile to estimate the error in transit time measurement. The flow is assumed to be laminar. The photons path is obtained from a Monte Carlo simulation. This is used to determine the maximum penetration depth of each diffusely reflected photon and therefore state how the recorded signal is composed of the signals originating from different depths to check the hypothesis that the error is systematically linked to the vessels diameter. A simplified geometry is set as a homogeneous layer structure of vessel wall, blood and vessel wall. The total thickness ranges from 1 mm to 5 mm. The probability density of the depth distribution of the diffusely reflected photons and the parabolic flow profile are convolved to obtain a weighted average of the ow velocity, which is set into relation with the mean ow velocity. The results show a clear dependency of the error in transit time measurement on the vessels diameter which complies qualitatively with literature and confirms the hypothesis.

Laser systems emitting radiation in the visible and near infrared region are potentially hazardous for the retina of the human eye. This can result in irreparable injuries due to photomechanical, photothermal or photochemical light-tissue interactions. This investigation focuses on the photothermal interaction for which a computer model is used to simulate the thermal behavior of the retina and to predict the injury threshold values. The most important factors are the wavelength of the radiation, the exposure time and the irradiance profile on the retina. For performing safety evaluations and classifications the laser safety standard IEC 60825-1:2014 has to be considered. These evaluations are based on emission limits which depend on the same above mentioned factors. According to the IEC 60825-1:2014, non-uniform retinal images are treated by an image analysis where an averaged spot size is used. This averaged size is calculated by the extent of the irradiance profile along two orthogonal directions. Unlike the laser safety standard, the computer model predicts the injury thresholds for an irradiance profile on the retina without averaging the spot size. In this investigation, a broad variety of non-uniform retinal images is investigated with regard to the injury thresholds predicted by the computer model and to the classifications according to the laser safety standard.

In this work, we present a brief overview of sapphire medical instruments. Sapphire demonstrates a unique combination of physical properties, such as high hardness and chemical inertness, biocompatibility and high thermal conductivity, high transparency in a wide spectral range that makes it suitable for various medical applications. We demonstrate the examples of scalpel, capillary needle for laser therapy, neuroprobe and applicator for cryosurgery. Each of them combines different modalities in one instrument. Among them are tissue resection, therapy via electromagnetic wave delivering, aspiration, diagnosis, and tissue freezing. Sapphire instruments can be accompanied with magnetic resonance imaging and allow multiple sterilization.

A term “theranostics” was coined more than two decades ago to tag an emerging paradigm of combining diagnostic and therapeutic capabilities for a modern personalized medicine. Biomedical applications of light also involve diagnostic (e.g., optical bioimaging) and therapeutic (e.g., light induced therapy) modalities, providing opportunity for the combined, theranostic approach. Optical transmission windows for biological tissues have been identified in near-infrared (NIR) region, allowing researchers to benefit from the reduced tissue scattering and autofluorescence in this spectral range and achieve optical imaging and imaging guided therapy of deeper tissues. NIR spectral region can also be beneficial for phototherapy (e.g., photodynamic therapy, PDT), allowing for deeper penetration of light to excite phototherapeutic agents. On the other hand, interaction of NIR light with tissues is known to produce beneficial therapeutic effects by itself: low level light therapy (a.k.a. photobiomodulation, PBM) with NIR light is reported to enhance wound healing, relieve pain, reduce inflammation and treat some pathological conditions.

Previous studies determining laser-induced damage thresholds show inconsistent injury results due to measurement techniques as well as methodological challenges using top hat profiles. One reason that decisively influences the ED₅₀ value is the appearance of so-called intensity modulations of a top hat associated with the quality of the energy uniformity on the irradiated tissue sample. By using two different square core fiber lengths (2 m, 20 m) to produce a top hat, we can on one hand ensure the focus control and the correlated homogenized intensity distribution and on the other hand vary and determine intensity modulations in the profile. This means that the accuracy of the measurements is significantly enhanced and the influence of energy variations in the profile can be evaluated explicitly and quantitatively. Our laser-induced irradiation experiments were performed with 1.8 ns pulses at a wavelength of 532nm on porcine RPE by means of a Q-switched, frequency-doubling Nd:YAG laser. Intensity modulations are characterized by a modulation factor that describes the ratio of maximum irradiation to the average. The generated factors in our measurements are 1.78 (high intensity variation) and 1.15 (low intensity variation). The calculated ED₅₀ values are 52.15 μJ for the higher modulations and 33.90 μJ for the lower modulations inducing a discrepancy factor of 1.54. Hence, the determined thresholds highlight the importance of the influence of intensity modulations within the beam profile during irradiation. Therefore, we examine this influence and its possible consideration and handling with determined threshold data. Furthermore, a strategy for ensuring a high top hat quality for the assessment of prospective threshold data is shown.

The ability to remotely extract temperature from a specific location using back scattered light analysis is very applicable. In this paper we present the first step towards remote sensing of temperature by using several approaches based upon conventional neural network analysis of the back scattered speckle patterns, analysis of the speckle patterns decorrelation time constant and Photoplethysmogram measurements.

Photoacoustic tomography is a new, rising imaging technique since it combines high penetration depth with good image contrast. Speckle-analysis represents an attractive, non-contact alternative to contact-based ultrasound transducers which are state of the art for photoacoustic tomography. In this work, it is demonstrated for the first time that the speckle-sensing technique is in theory capable of photoacoustic image reconstruction by measuring on several surface locations after photoacoustic excitation. Therefore, a simulative model is used to test a back-reconstruction algorithm and a first feasibility experiment is shown.

Tissue classification interest is growing due to their numerous applications in the biomedical framework. This increasing interest leads to the rise of alternative techniques in order to enhance the classification accuracy. Polarimetric methods, and in particular, the study of depolarization properties of samples, appear to be an alternative and complementary technique that helps to discriminate between different organic tissues. In fact, depolarization is directly related with the scattering processes produced in biological tissues, providing different responses when studied in different fibre based structures. In the current state of art, there exists different metrics devised to study depolarizing properties of samples and it is not clear which method is the most suitable for tissue classification. By using some of those polarimetric metrics, a compendium of depolarizing spaces can be constructed, composed of three observables each. Such depolarizing spaces stand out over the other existing polarimetric indicators as they allow synthetizing the depolarization content of samples, to provide further analysis of depolarizers. In this work, we compare the effectivity of different depolarizing spaces in order to determine the best space for tissue classification. This is done by using a collection of chicken thigh tissues. In particular, we have measured the Mueller matrix of 120 samples spread around three chicken thigh tissues: tendon, muscle and myotendinous junction. Different tissues have been imaged at different wavelengths (470 nm, 530 nm and 625 nm) to obtain more information and to amplifying the classification sensitivity with this extra channel (wavelength). Finally, we conclude the work by showing the depolarization space providing the best results in terms of tissue classification.

To fulfill continuous blood pressure measurement, we developed a cuff-less blood pressure (BP) monitoring method which was based on the pulse transit time (PTT). We measured photoplethysmography (PPG) signals at two different positions simultaneously to solve the disadvantages of the cuff-based BP measurement, which was unsuitable for those had stiff artery issues or atrial fibrillation problems. Therefore, we replaced the inflatable cuff with two PPG pods which required just simple contact to the skin surface. With two optical pods being placed at two different positions, the PTT was calculated from the PPG waveforms. The time difference between the R-peak of an electrocardiogram (ECG) and the PPG foot point was traditionally taken as the PTT parameter for blood pressure model calculation. We replaced the calculation between the ECG and the PPG signals by detecting two PPG waveforms at the same time. The relationship between the PTT and BP (including systolic blood pressure (SBP) and diastolic blood pressure (DBP)) has been investigated and shown with high correlation.

Early diagnostics of microcirculation complications is an important area for biomedical photonics application. The blood perfusion measurements are capable of identification of particular markers for diagnostics of many pathological conditions of blood microcirculation in the skin. In this work, we apply the laser Doppler flowmetry method with the ability to record and process the power spectra of registered photocurrent. This approach provides the estimation of signal amplitude distribution along with the frequencies of Doppler-broadened laser radiation and blood perfusion distribution. In this work, we investigate the blood ow in the skin by the time- frequency analysis of the recorded laser Doppler spectra. The conducted studies allowed us to propose new diagnostic criteria for the diagnosis of diabetes mellitus type 2 complications. The diagnostic parameters have been tested together with binary classifiers based on the linear discriminant analysis and demonstrated to be able to successfully distinguish the groups of volunteers of different age and patients with microvascular complications.

The article is aimed at conducting pilot studies of microcirculation changes in patients with diabetes mellitus during the course of intravenous infusions of alpha lipoic acid. The study was conducted with patients with diabetes during the passage of a standard course of treatment. The change in parameters was compared before the start of the infusion course, during and after the course. A system of wearable laser Doppler monitors was used to conduct the study. The study showed that although the microcirculation index does not undergo significant changes during the treatment process, spectral analysis of the recorded signal has potential applicability. Further studies with a larger sample group are required for a qualitative analysis of the observed effects.

Most cervical cancers originate from the epithelial layer by an uncontrolled growth of abnormal squamous cells and are known as carcinomas. Early stages of this disease manifest as biochemical and morphological changes in the superficial layer. Such changes can be captured from the spectral behavior of intrinsic fluorophores present in the layered cervical tissue. Fluorescence spectroscopy is thus widely used for detection of pre-cancers, also due to its capability as a fast, non-invasive and quantitative probe. This study focuses on analysis of the spectral information of the fluorophores using spatially resolved fluorescence spectroscopy for diagnosis of cervical cancer at an early stage. An in-house fabricated fiber-optic probe of diameter 1mm, consisting of 77 fibers in approximately five circular rings with very high sensitivity for superficial layer changes, has been used to collect fluorescence spectra from different spatially resolved positions of two layered solid phantoms. The phantoms are prepared by varying the thickness and fluorophore concentration of the upper layer. Optical properties of these layered phantoms have been kept similar to cervical tissue to replicate the subtle changes that occur in the tissue with the growth of abnormality. A 405 nm laser diode source is used to excite the samples with two different fluorophores in the two layers, Flavin Adenine Dinucleotide (FAD) in upper layer and Proto-porphyrin (PpIX) in bottom layer. A `Look-up Table' method is used to finally reconstruct thickness and fluorophore concentrations of upper layer of an unknown phantom by evaluating the peak ratios of fluorophores from spectra obtained at different spatially resolved positions.

The possibility of using a multi-modal approach in the clinical practice of a dermatologist for psoriasis, which includes 3 optical non-invasive technologies: laser Doppler flowmetry, video capillaroscopy, and laser speckle-contrast imaging, is demonstrated. The dynamics of changes in the main microhemodynamic parameters of the microcirculatory bed of patients with psoriasis during conservative therapy was studied, and the effectiveness of treatment of patients suffering from psoriasis was evaluated.

In the paper, we introduce an additive simulation approach of Raman light scattering by skin cancer using the Monte Carlo method. Raman light scattering from normal skin and malignant melanoma is investigated. A two-stage algorithm for simulating Raman light scattering from skin based on the known photon transport algorithm has been developed. A method for additive modeling of skin pathologies is proposed. The main idea of this method is a hypothesis that an experimental Raman spectrum of normal skin, obtained by averaging in vivo Raman spectra of normal skin, may be served as a “substrate” for the feature simulated Raman spectrum. Thus, the pathology, for their part, may be “grown” by adding on this “substrate” Raman specific components set related to a tumor type. Additive simulation of malignant melanoma has been carried out. The possibility of using the developed algorithm to determine the component composition of the skin by the in vivo Raman spectrum of skin is discussed. An attempt to evaluate the change in the concentration of skin components during the development of cancer has been made.

A method for formation of bionanocomposites that can be used as coatings for implantable devices interacting with blood is proposed. Such bionanocomposites are composed of albumin blood protein and single-walled carbon nanotubes (SWCNT). They are formed by evaporation of water-albumin dispersion of nanotubes by diode laser with precise temperature control. In this work influence of various types of SWCNT graphene structure defects on formation of frame structure from them and albumin was determined using Raman and FTIR spectroscopy methods. It was found that shape of graphene sheet affects the intensity of the D and D’ bands in its Raman spectrum. Strongest increase in the intensity of D band compared to G band is observed in case of partial or complete absence of hydrogen atoms at the boundary of graphene sheet, which leads to practical disappearance of G and D’ lines in Raman spectrum. Molecular modeling showed that for structural defects of graphene sheet edge there is a steric hindrance, which occurs when vibrations of neighboring hydrogen atoms moving towards each other. Possibility of SWCNT functionalization by oxygen atoms of negative amino acid residues aspartic (Asp) and glutamic (Glu), which are located on the outer surface of BSA, is demonstrated. Formation of covalent bonds between BSA and SWCNTs is confirmed by FTIR and Raman spectra. Spectra were recorded for small diameter nanotubes (1.7 nm) with various concentrations (0.01, 0.1, and 1 g/mol). An increase in concentration of nanotubes leads to saturation of functionalization of SWCNTs with oxygen atoms of amino acid residues Asp and Glu. Covalent interaction of BSA with SWCNTs disrupts secondary and tertiary albumin structure. This is confirmed by significant decrease in absorption bands intensity in high-frequency region. Formation of covalent bond between BSA and SWCNTs in composite leads to an increase in band intensity in ~1330 cm^-1 region on Raman spectrum compared to the spectrum of initial nanotubes. This confirms presence of significant defects in SWCNTs caused by covalent addition of oxygen to graphene surface of nanotubes. It was found that increase in diameter of nanotubes (4 nm) and their concentration in composite practically does not affect vibrational spectra, which confirms the hydrophobic interaction of BSA and SWCNTs. Thus, two types of interactions in solid composites based on BSA with SWCNT – hydrophobic and with formation of covalent bonds – depend on the diameter of nanotubes used. This criterion makes it possible to widely use frame composites to control the state of blood cells aggregation.

The results of an experimental study of changes in the microcirculation of subcutaneous blood flow in a malignant tumor using laser speckle contrast imaging (LSCI) using an optical clearing agent of 70% glycerol solution are presented. In this study, for hemodynamic monitoring we used the speckle contrast method, which is one of the laser speckle imaging methods. This method is based on calculation of contrast of time-averaged dynamic speckles in dependence on the exposure time at the registration of the speckle-modulated images. Analysis of change of hemodynamic was carried out depending on the stage of development of pathologyIn research, we used 15 white laboratory rats Wistar weighing 300-500 g. A tumor was inoculated in laboratory rats by subcutaneous injection of 0.5 ml of a 25% tumor suspension into the area of the shoulder blades in a Hanks solution strain of PC1 alveolar liver cancer. Laboratory animals were divided into three groups, the first included healthy animals, the second - animals with an inoculated tumor 14 days after injection and the third - animals with an inoculated tumor 28 days after injection. It was found that the blood flow rate in pathological group No. 2 was reduced by 25% relative to control measurements of group No. 1, in pathological group No. 3 increased by 20% relative to control measurements. Also, the results showed that the use of a 70% glycerol solution reduced the blood flow velocity in the control group by about 2 times. Moreover, in pathological groups, the effect of the solution on the blood flow is more pronounced, in the group, the measurements on which were carried out 14 days after the inoculation of the tumor, the blood flow rate decreased by 2.8 times, after 28 days - by 4 times. The results obtained in the study of disorders of the microcirculation of the subcutaneous blood flow under the conditions of the development of a malignant tumor show that the development of diseases in animals causes changes in the microcirculatory system, and the use of optical clearing agents demonstrates changes in vascular permeability under conditions of the development of pathologies.

Breast cancer arises either in the lobules or the ducts of the tissue. Structural changes occurring with malignancy manifest as refractive index variations inside the tissue. It is crucial to quantify these depth wise variation in refractive index for early cancer detection. In this study, three types of unstained breast tissue sections - fibrocystic, fibroadenoma, and invasive carcinoma have been examined for the ultra-structural changes using `Fourier domain low coherence interferometry'. The resulting interference spectra of the backscattered light from the front and the rear surface of the sample are Fourier analyzed to provide depth correlation function. The subtle small-scale fluctuations in the Fourier analyzed spectra are then evaluated using Discrete Wavelet Transform (DWT). Daubechies-1 wavelet of DWT is used to calculate the high pass and low pass coefficients. The sixth level low pass coefficients of DWT clearly discriminate among normal, benign, and malignant breast tissue. Skewness and kurtosis values for these coefficients are also able to well distinguish the type of tissues.

Spinal cord injury (SCI) occurs as a result of damage to neurons in the spinal cord, which causes an interruption of neural signal conduction along the axonal tracts. SCI can result in complete or partial paralysis. Several studies have demonstrated that photobiomodulation therapy (PBMT) has the ability to repair nerves and enhance functionality. PBMT is also known to present a biphasic dose-response pattern. Various PBMT research studies on contusion spinal cord injuries in rats have applied a fixed fluence onto the rats’ skin during the study period without considering the rats’ weight, which can lead to a reduction of the delivered fluence delivered to the SCI site. Therefore, the purpose of this work is to evaluate whether the bodyweight of the rat model, as well as the irradiation parameter, will in fact affect the fluence delivered to the SCI site. The study employed four computational rat models of the same age (11 weeks) and four different weights (250, 270, 290, and 310 g) as well as different irradiation parameters (3 wavelengths ×15 beam diameters × 2 beam shapes) to study their effect on the fluence delivered to the SCI site using Monte Carlo simulation. As the rat’s weight during the study period decreased, more fluence was delivered to the SCI site. Our results showed that the percentage increase of the fluence delivered to the SCI site was highest for the 660 nm large gaussian beam when compared with other parameters. We found that the irradiation parameters and the rats’ weight strongly influenced the dosage delivered to the SCI site in the rat. Therefore, researchers should consider the fluence delivered to the injury site instead of onto the skin when studying the effectiveness of PBMT.

The digital diaphanoscopy method seems promising for solving one of the urgent problems of modern otolaryngology, which is associated with providing accurate, painless and timely diagnosis of pathologies of the maxillary sinuses. Optical properties of the study area and their changes for different anatomical and gender features and pathologies presence determine the results of digital diaphanoscopy. Adjusting the parameters of the probing and measuring parts of digital diaphanoscopy devices taking into account these factors is important task to obtain similar light scattering patterns for different patients and the possibility of their subsequent comparison. This paper presents the improved numerical model of the maxillary sinuses. The developed model considered the maxillary sinuses sizes, their location and asymmetry, various thicknesses of bone and soft tissues, size and localization of pathology (cystic fluid and tumor). The cross-sectional face model was more detailed and considered the curvature of the face. Simulation was carried out at various positioning of radiation source relative to the study area and detector. Additionally, the various rotation angles of the study area with radiation source relative to the detector were considered. The attenuation of probe radiation intensity at the detector was estimated using the Monte Carlo method in the TracePro (Lambda Software) for visible and near-IR wavelengths at the different intensity values. The correlations between model signal and anatomical features of the study area, and the changes of the study area position were identified. It was established that registration of light scattering patterns at different position is important parameter for more accurate evaluation of the maxillary sinuses state and the localization of pathologies. The threshold values of the probe radiation intensity and the optimal study positions, which provide the optimal signal-to-noise ratio of the detectable signals, were identify.

Temperature dependences of the spectral characteristics of the rat skin, muscle and fat tissues were obtained in vitro. The changes in the spectral characteristics of the rat skin subjected to 60 min thermal treatment at elevated (>40°C) temperatures have been characterized. The dynamics of the changes in collimated transmittance of rat skin at elevated temperature is shown to be temperature-dependent. It is assumed that the changes in spectral characteristics might be caused by changes in light scattering regime within the sample.

In this paper, we assess the quality of three speckle image processing techniques - namely Laser Speckle Contrast Imaging (LSCI), Fuji method and Generalised Differences (GD) - in the detection of veins in various plant leaves. We develop a simple NIR laser speckle imaging set-up and find that the Fuji algorithm is best suited for producing highly contrasted and spatially resolved veins in leaf tissue, with the LSCI algorithm producing lower resolution results and GD failing to locate fine vascular structure. Determining the practicality of various speckle image processing techniques for vein detection aides future research for possible subcutaneous blood ow detection in non-invasive biomedical applications.

In this study a hand held probe has been automated using a motorized stage and LabVIEW based control and acquisition system for in-vitro cervical precancer diagnosis. The stage movement is controlled by two motors for vertical and horizontal movement while a third motor is utilized for changing the polarization state. The data acquisition and storing was thus performed by moving the probe to a specific site, acquiring polarized fluorescence and elastic scattering signals and creating a user friendly graphical user interface(GUI) panel to suit our needs. Another GUI in MATLAB has also been developed for the real time analysis of data captured through the automatic acquisition system. Three windows were created inside the GUI to display calculated intrinsic fluorescence, for statistical analysis and classification. Principal component analysis was applied to extract the features and classification was done by Mahalanobis distance based algorithm. The classification results have been presented of different grades of cervical cancer

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.

An experimental method for remitted photon path length measurements in scattering media has been developed and tested on human skin and skin neoplasms, skin phantoms and cell cultures. The photon time-of-flight (PTOF) measurement method was used in this study, where the photon travel time was converted into path length. Remitted light signals were obtained using a picosecond broadband laser and a set of narrowband interference filters in spectral rang 520 – 760 nm. Five different distances of 1, 8, 12, 16 and 20 mm between the source and detector fibers were used. Measurements were performed at different wavelengths and distance combinations; they were taken from human skin and skin malformations, agar-based phantoms with different concentrations of intralipid and hemoglobin, and from cell cultures (DC3F, B16/F10). Parameters related to the remitted photon mean path length will be presented and analyzed.

The problem of diabetes mellitus (DM) has attracted scientists of different specialities, as its prevalence is increasing worldwide and assumes the character of a pandemic. The use of optical non-invasive diagnostic methods is a promising direction for the early determination of the presence and severity of diabetic complications. This work is aimed at assessing changes in the uorescence intensity and the blood microcirculation level, evaluated in the skin of patients with DM with the use of local cold and heating tests. An experimental studies were carried out involving patients with type 2 DM and healthy controls. Significant differences were found in the changes in the level of microcirculation and oxidative metabolism under the in uence of local temperature effects (local heating and cooling) among patients and control group. The results can be used in further development of an optical non-invasive diagnosis of diabetes complications.

Fiber-optic probes are widely used in biomedical research as convenient and compact devices for instrument-tissue interaction analysis. This paper presents a simulation of various configurations of fiber optic probes that implement selective registration of the spectral response from skin layers. The impact of the registration channel of the optical scheme on the skin fluorescence spectrum was analyzed upon excitation by laser radiation with a wavelength of 405 nm. A model of human skin has been developed, containing the main endogenous chromophores of the skin for a specific excitation wavelength. We compared the model skin fluorescence spectra with experimental spectra from real tissue. The propagation of excitation and fluorescence radiation in the system was simulated by the Monte Carlo method. It has been observed that it is necessary to take into account the optical design and the efficiency of collecting fiber probe in examining the spectral response from a multilayer biological tissue such as human skin. Because for fixed parameters of the tissue site under investigation, the recorded fluorescence spectrum at the detector varies depending on the sensitivity of the probe to individual layers of the biological tissue.

The new generations of photoactive compounds, which are derivatives of already well-established groups of photosensitizers, together with the modern light sources in the spectral region of their activation and in the PDT window range, are emerging as a reliable phototherapy approach for tumour treatment with higher efficiency. The substitution of a highly hydrophobic phthalocyanine with galactose units could improve the solubility and to decrease significant aggregation in polar solvents. The control group of rats with transplanted cholangiocarcinoma without any treatment was used for comparison of PDT effectiveness. In the second group the white outbred male rats with cholangiocarcinoma lesions were treated using Zn-phthalocyanine, as a typical representative of phthalocyanine photosensitizers with known photodynamic properties and the third group of animals was treated with galactose – Lu-phthalocyanine, as a novel derivative from the same family with two specific differences – presence of galactose and replacing of Zn(II) with Lu(III) ion. To compare the PDT effectiveness of both compounds the same drug doses were applied - 2 mg/kg, applied by intratumoural injection. Diode laser source at 670 nm, on 50 mW output power with power density applied 200 mW/cm2 was applied for 1000 sec to obtain total irradiation dose of 200 J/cm2 for each lesion. 3 days after treatment, the animals were withdrawn from the experiment. Tissue from the central zone and periphery of the tumor was taken for morphological examination and fixed in 10% formalin. Serial paraffin sections were stained with hematoxilin-eosin and by immunohistochemical staining with antibodies to proliferation marker Ki-67 and apoptosis marker BAX. After photodynamic therapy, pronounced necrobiotic changes in combination with an inflammatory reaction were developed at the central zone of tumors. The dystrophic changes, reduction of proliferation and development of apoptosis in tumor cells were observed on tumor periphery as well.

Liposomal particles are used as an instrument for drug delivery and as fluorescent labels. Due to its properties, liposomal particles can be used as a dual-purpose contrast agent both in fluorescence imaging, laser speckle contrast imaging and ultrasound diagnostics. Liposomes obtained by one purpose but subsequently contained a fluorescent label or air. This work aimed to prepare liposomal particles and to study possibilities of its application for the dual-use in fluorescence spectroscopy and ultrasound diagnostics. In this work, we used fluorescence spectroscopy to investigate the efficiency of propagation in the circulatory system of fluorescent-labelled liposome nanocapsules loaded with eosin-Y (disodium salt of 2,4,5,7 tetrabromofluorescein These results demonstrate that liposomal particles can be used in fluorescence spectroscopy, in ultrasound diagnostics and in LSCI. It may find the application in the field of drug delivery control and high-throughput screening during drug development.

Solving the problem of limited depth penetration of light in vivo remains important due to the innate high scattering of light in the tissues. One way to reduce tissue scattering is to apply immersion optical clearing using chemical agents. This reversible process is based on partial replacement of tissue intercellular fluid with clearing agents, which leads to matching of refractive indices between tissue components as well as tissue dehydration and consequently to more regular packing of tissue structural elements. Earlier we discovered that low molecular weight MRI contrast agents have the ability of optical clearing [1]. This was shown for the first time in the case of mouse skin samples ex vivo. The next logical step included an in vivo study. The expected advantages of using MRI contrast agent compositions as optical clearing agents are: 1) their biocompatibility; 2) the possibility of combining the areas of enhanced contrast of MRI and optical images in time and space. The effect of optical clearing was obtained when MRI contrast agents were applied to the surface of mouse skin in vivo. We used the following MRI contrast agents: Gadavist (Bayer HealthCare Pharmaceuticals, Germany), Magnevist (Bayer HealthCare Pharmaceuticals, Germany) and Dotarem (Guerbet, France). The Spectral Radar OCT System OCP930SR 022 (Thorlabs Inc., United States) with a wavelength of 930 nm was used to quantify the change in the optical properties of the skin. After hair removal from the mouse skin, a B-scan of the intact skin region with OCT was recorded. Then, the selected MRI agent was applied topically to the target skin area. Skin OCT scans were recorded every 5-10 min during the exposure of the skin area to an MRI agent. The solution was removed each time before scanning and applied again after scanning. OCT showed an increase in the penetration depth of the light beam into the tissue and resulted in more contrast images of the skin by reducing skin scattering for each of the used MRI agents. OCT data were processed to quantify the diffusion coefficients of MRI agents in the skin and the effectiveness of optical clearing of skin using these agents. The obtained results show the possibility of not only improving the quality of optical imaging, but also open the way for the implementation of a new approach to multimodality, when synchronization in time and space of regions with increased contrast of both the optical image and MRI image is automatically ensured since the same agent enables both effects simultaneously.

Application of many optical methods, such as photoacoustics, optical coherent tomography, and terahertz spectroscopy, for tissue imaging and disease diagnostics is limited due to small probing depth caused by strong scattering and absorption in tissues [1, 2]. It is possible to increase the efficiency of optical techniques by using tissue optical clearing (TOC), which allows one to control tissue optical properties by exposing tissue to biocompatible liquids - optical clearing agents (OCAs). When exposed to an OCA with hyperosmotic properties, such as glycerol or highly concentrated glucose solution, it is possible to achieve a considerable decrease of tissue scattering within the entire spectral range and absorption on the water bands due to refractive index matching phenomenon and dehydration of the tissue, and therefore to get a significant increase of the optical and THz wave transmittance [1-3]. In vitro studies were performed on laboratory rats of the Vistar strain (sexually mature females weighing 300–400 g were used). The animals were inoculated with the tumor cells by subcutaneous injection into the area of the scapulae. Studies were conducted in 28 days after injection. The weight and thickness of the samples were measured before and after optical clearing by using a micrometer with an accuracy of ±0.001 mm and an analytical balance (Scientech, SA210, USA) with an accuracy of ±1 mg. The samples of the area 1.5*1.5 cm2 were placed in a closed sealed container, to which was added 2 ml of OCA. 70% glycerol solution, 40% glucose solution and 50% fructose in a 30% alcohol solution were used with the addition of saline as an OCA. The transmission spectrum of glycerol in the frequency range of 0.05–1.0 THz was measured by using the terahertz (THz) time-domain spectrometer. Measurements of the refractive index of OCA were carried out before the start of the experiment and every 5 minutes after placing the skin sample in the solution with OCA for 15 minutes, then every 10 minutes for another 45 minutes. It was found that the effectiveness of optical clearing of the skin over the tumor is higher compared to healthy skin. A good correlation was shown between the kinetics of the sample mass and THz absorption and refraction. An assessment of skin dehydration also showed that the concentration of water extracted from the skin above the tumor is greater compared to healthy. It is especially important that enhancing the image contrast due to dehydration and TOC in the pathology area is extremely significant in diagnosing precancerous conditions and treating early stages of tumor, due to the possibility of selective increase of the scattering contrast of cell nuclei relative to surrounding cytoplasm and interstitial fluid and thus following their size and internal rearrangements [5].

Photosensitizers (porphyrins) attack cancer cells and during photoactivation contribute to the generation of reactive oxygen species that damage various structures of cancer cells, which leads to their destruction. Porphyrins reach tumors through the blood and modelling of conditions for the transfer of porphyrins to the tumor (binding to blood proteins, the influence of pH and salt composition of the medium) is one of the most important problem of photodynamic therapy (PDT) of tumor. Ceruloplasmin (CP) is an important copper-containing plasma protein that actively binds photosensitizers (PS) and can play a certain role in the transfer of photosensitizers to the tumor. Changes in pH, as well as of salt composition in the tumor tissue and its cells can cause modifications in the complexes [CP + PS] and a possible separation of the PS from the complex can change the photodynamic process of tumor destruction. In this paper, it was shown that: 1) with a change in the pH of the medium from neutral to acidic, as well as with a change in the salt composition of the medium, significant conformational changes of ceruloplasmin and the transition of photosensitizers to the surface (for cationic porphyrins) or to the inside of the protein macromolecule (for chlorin e₆) occur, with a partial separation of photosensitizers from protein; 2) CP can form complexes with photosensitizers and be an active carrier of PSs in the blood.

The results of expanded spectral analysis of donor bone implants using the Raman spectroscopy method are presented in the work. Mathematical methods of improving resolution of spectral contours and chemometric analysis are used for evaluation of component composition of the implants. It is shown that the Raman spectroscopy can be used for evaluation of relative concentration of mineral and organic components in extracellular matrix of bone tissue. Spectral features of the samples of bone tissues demineralized and processed with ultrasound with different degree are found as a result of the study. The criteria allowing evaluating the component composition during manufacture of bioimplants are suggested in the work.

The results of research of samples of distal femoral epiphysis in rabbits that had different types of plasty using the Raman spectroscopy method are presented. The component analysis of the samples under study has been made using spectra deconvolution by spectral contour selection The efficiency of the method of treatment of cartilage destruction using the combination of platelet-rich plasma with a bone is shown. It has been established that Raman spectroscopy can be used for estimation of areas of articular surface after chondroplasty and of degree of cartilage recovery.

The results of experimental study of samples of dental tissues affected by periodontitis using Raman spectroscopy method are presented in the work. Spectral changes are detected in hard dental tissues affected by periodontitis. The main spectral changes are seen in the wavenumbers of 956 cm^-1, 1236 cm^-1, 1269cm^-1 and 1663 cm^-1, owing to the change of mineral-protein components in case of periodontitis.

Stimulated emission depletion (STED) nanoscopy is a typical super-resolution imaging technique that has become a powerful tool for visualizing intracellular structures on the nanometer scale. Aggregation-induced emission luminogens (AIEgens) are ideal fluorescent agents for bioimaging and have been widely used for organelle targeting, cellular mapping and tracing. Since AIEgens generally have a large Stokes’ shift, which is beneficial for restraining the fluorescence background induced by the STED light, as well as high photobleaching resistance in their nanoaggregate states, which provides the potential for long-term imaging under a STED beam with high power density, they are ideal fluorescent agents for STED nanoscopy. The STED efficiency of aggregated TPA-T-CyP could reach more than 80%, and the dynamic mitochondrial visualization was achieved on the nanometer scale. Their moving, fission and fussion of mitochondria was clearly observed with a lateral spatial resolution of 74 nm.