Detection of NADH and NADPH levels in vivo identifies shift of glucose metabolism in cancer to energy production.

Profound changes in the metabolism of cancer cells have been known for almost 100 years, and many aspects of these changes have continued to be actively studied and discussed. Differences in the results of various studies can be explained by the diversity of tumours, which have differing processes of energy metabolism, and by limitations in the methods used. Here, using fluorescence lifetime needle optical biopsy in a hepatocellular carcinoma (HCC) mouse model and patients with HCC, we measured reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in control liver, and in HCC tumours and their adjacent regions. We found that NADH level (mostly responsible for energy metabolism) is increased in tumours but also in adjacent regions of the same liver. NADPH level is significantly decreased in the tumours of patients but increased in the HCC mouse model. However, in the ex vivo tumour slices of mouse HCC, reactive oxygen species production and glutathione level (both dependent on NADPH) were significantly suppressed. Thus, glucose-dependent NADH and NADPH production in tumours changed but with a more pronounced shift to energy production (NADH), rather than NADPH synthesis for redox balance.

Incremental residual polarization caused by aging in human skin.

Significance: The study of the effect of aging on the optical properties of biological tissues, in particular polarization, is important in the development of new diagnostic approaches. Aim: This work aims to provide a comprehensive analysis of the factors and mechanisms that contribute to the alteration of skin polarization properties caused by aging, using polarization-sensitive hyperspectral imaging measurements and Monte Carlo simulation. Approach: Our investigation involved both experimental studies of in vivo human skin of volunteers of different ages and computational modeling that accounted for changes in the absorption and scattering properties of the skin model. Specifically, we analyzed alterations in the degree of linear polarization (DOLP) to better understand the impact of aging on skin polarization properties. Results: A statistically significant increase in the DOLP was found for the elderly group. At the same time, there was no correlation between changes in polarization and the calculated blood volume fraction parameter for different ages. According to the simulation results, it was also found that a change in the scattering properties of biological tissues has a more significant effect on the change in polarizing light compared to the change in absorption. Conclusions: The results of the work prove that the sensitivity of polarization imaging to age- or pathological-related skin changes may be primarily due to changes in scattering, which in turn is associated with changes in the collagen fibers of the dermis. The proposed technique shows promise for in vivo non-invasive real-time assessment of age-associated skin changes and can also be extended to monitor changes associated with the development of age-related pathologies.

Efficiency of direct photoinduced generation of singlet oxygen at different wavelengths, power density and exposure time of laser irradiation.

This work investigates the influence of laser irradiation parameters (wavelength, power density and exposure time) on singlet oxygen (1O2) generation efficiency. Chemical trap (L-histidine) and fluorescent probe (Singlet Oxygen Sensor Green, SOSG) detection methods were used. Studies have been conducted for 1267, 1244, 1122 and 1064 nm laser wavelengths. 1267 nm had the highest efficiency of 1O2 generation, but 1064 nm demonstrated almost the same efficiency. We also observed that the 1244 nm wavelength can generate some amount of 1O2. It was demonstrated that laser exposure time can generate 1O2 more efficiently than an increase of power. Additionally, the SOSG fluorescence intensity measurements method for acute brain slices was studied. This allowed us to evaluate the approach's potential for in vivo detection of 1O2 concentrations.

Sampling volume assessment for wearable multimodal optical diagnostic device.

The process and results of numerical Monte Carlo simulation of optical radiation propagation in laser Doppler flowmetry (LDF) and fluorescence spectroscopy (FS) channels of a wearable diagnostic multimodal device are described in this paper. To achieve the goal, a multilayer skin model with different parameters of blood and melanin content and different distances between sources and radiation receivers was designed. The changes in the sampling (diagnostic) volume depending on the anatomical features of the biological tissues, as well as on the technical parameters of the device were shown. Depending on the scattering media optical properties and the source-detector configuration of the device, the diagnostic volume can range from 2 to 7 mm3 . The obtained results allow the formation of specialized medical and technical requirements for wearable multimodal devices implementing LDF and FS channels.

Digital diaphanoscopy of maxillary sinus pathologies supported by machine learning.

Maxillary sinus pathologies remain among the most common ENT diseases requiring timely diagnosis for successful treatment. Standard ENT inspection approaches indicate low sensitivity in detecting maxillary sinus pathologies. In this paper, we report on capabilities of digital diaphanoscopy combined with machine learning tools in the detection of such pathologies. We provide a comparative analysis of two machine learning approaches applied to digital diapahnoscopy data, namely, convolutional neural networks and linear discriminant analysis. The sensitivity and specificity values obtained for both employed approaches exceed the reported accuracy indicators for traditional screening diagnosis methods (such as nasal endoscopy or ultrasound), suggesting the prospects of their usage for screening maxillary sinuses alterations. The analysis of the obtained values showed that the linear discriminant analysis, being a simpler approach as compared to neural networks, allows one to detect the maxillary sinus pathologies with the sensitivity and specificity of 0.88 and 0.98, respectively.

Lesion Detection and Analysis Using Optical Imaging.

The biomedical application of optical spectroscopy and imaging is currently an active, developing area of research, supported by recent technical progress in the development of light sources and detectors [...].

Diagnosis of Skin Vascular Complications Revealed by Time-Frequency Analysis and Laser Doppler Spectrum Decomposition.

Nowadays, photonics-based techniques are used extensively in various applications, including functional clinical diagnosis, progress monitoring in treatment, and provision of metrological control. In fact, in the frame of practical implementation of optical methods, such as laser Doppler flowmetry (LDF), the qualitative interpretation and quantitative assessment of the detected signal remains vital and urgently required. In the conventional LDF approach, the key measured parameters, index of microcirculation and perfusion rate, are proportional to an averaged concentration of red blood cells (RBC) and their average velocity within a diagnostic volume. These quantities compose mixed signals from different vascular beds with a range of blood flow velocities and are typically expressed in relative units. In the current paper we introduce a new signal processing approach for the decomposition of LDF power spectra in terms of ranging blood flow distribution by frequency series. The developed approach was validated in standard occlusion tests conducted on healthy volunteers, and applied to investigate the influence of local pressure rendered by a probe on the surface of the skin. Finally, in limited clinical trials, we demonstrate that the approach can significantly improve the diagnostic accuracy of detection of microvascular changes in the skin of the feet in patients with Diabetes Mellitus type 2, as well as age-specific changes. The results obtained show that the developed approach of LDF signal decomposition provides essential new information about blood flow and blood microcirculation and has great potential in the diagnosis of vascular complications associated with various diseases.

Simulation of thermal field distribution in biological tissue and cell culture media irradiated with infrared wavelengths.

In recent years, there has been a growing interest in the singlet form of oxygen as a regulator of the physiological functions of cells. One of the ways to generate singlet oxygen is direct optical excitation of the triplet oxygen form. Since molecular oxygen weakly absorbs light, high power is required to obtain sufficient concentrations of singlet oxygen. However, the increase in the radiation power of laser can induce a local temperature increase around the laser spot. This may be critical considering the temperature governs every biological reaction within living cells, in particular. Here, the interaction of laser radiation of infrared wavelengths, generating singlet oxygen, with biological tissues and cell culture media was simulated. Using the COMSOL Multiphysics software, the thermal field distribution in the volume of skin, brain tissue and cell culture media was obtained depending on the wavelength, power and exposure time. The results demonstrate the importance of taking temperature into account when conducting experimental studies at the cellular and organismal levels.

Laser-induced singlet oxygen selectively triggers oscillatory mitochondrial permeability transition and apoptosis in melanoma cell lines.

Singlet oxygen (1O2) is an electronically excited state of triplet oxygen which is less stable than molecular oxygen in the electronic ground state and produced by photochemical, thermal, chemical, or enzymatic activation of O2. Although the role of singlet oxygen in biology and medicine was intensively studied with photosensitisers, using of these compounds is limited due to toxicity and lack of selectivity. We generated singlet oxygen in the skin fibroblasts and melanoma cell lines by 1267 nm laser irradiation. It did not induce production of superoxide anion, hydrogen peroxide or activation of lipid peroxidation in these cells confirming high selectivity of 1267 nm laser to singlet oxygen. 1O2 did not change mitochondrial membrane potential (ΔΨm) in skin fibroblasts but induced fluctuation in ΔΨm and complete mitochondrial depolarisation due to opening permeability transition pore in B16 melanoma cells. 1267 nm irradiation did not change the percentage of fibroblasts with necrosis but significantly increased the number of B16 melanoma cells with apoptosis. Thus, singlet oxygen can induce apoptosis in cancer B16 melanoma cells by opening of mitochondrial permeability transition pore (PTP) but not in control fibroblasts.

Fluorescence lifetime needle optical biopsy discriminates hepatocellular carcinoma.

This work presents results of in vivo and in situ measurements of hepatocellular carcinoma by a developed optical biopsy system. Here, we describe the technical details of the implementation of fluorescence lifetime and diffuse reflectance measurements by the system, equipped with an original needle optical probe, compatible with the 17.5G biopsy needle standard. The fluorescence lifetime measurements observed by the setup were verified in fresh solutions of NADH and FAD++, and then applied in a murine model for the characterisation of inoculated hepatocellular carcinoma (HCC) and adjacent liver tissue. The technique, applied in vivo and in situ and supplemented by measurements of blood oxygen saturation, made it possible to reveal statistically significant transformation in the set of measured parameters linked with the cellular pools of NADH and NADPH. In the animal model, we demonstrate that the characteristic changes in registered fluorescent parameters can be used to reliably distinguish the HCC tissue, liver tissue in the control, and the metabolically changed liver tissues of animals with the developed HCC tumour. For further transition to clinical applications, the optical biopsy system was tested during the routing procedure of the PNB in humans with suspected HCC. The comparison of the data from murine and human HCC tissues suggests that the tested animal model is generally representative in the sense of the registered fluorescence lifetime parameters, while statistically significant differences between their absolute values can still be observed.

Interaction of Mitochondrial Calcium and ROS in Neurodegeneration.

Neurodegenerative disorders are currently incurable devastating diseases which are characterized by the slow and progressive loss of neurons in specific brain regions. Progress in the investigation of the mechanisms of these disorders helped to identify a number of genes associated with familial forms of these diseases and a number of toxins and risk factors which trigger sporadic and toxic forms of these diseases. Recently, some similarities in the mechanisms of neurodegenerative diseases were identified, including the involvement of mitochondria, oxidative stress, and the abnormality of Ca2+ signaling in neurons and astrocytes. Thus, mitochondria produce reactive oxygen species during metabolism which play a further role in redox signaling, but this may also act as an additional trigger for abnormal mitochondrial calcium handling, resulting in mitochondrial calcium overload. Combinations of these factors can be the trigger of neuronal cell death in some pathologies. Here, we review the latest literature on the crosstalk of reactive oxygen species and Ca2+ in brain mitochondria in physiology and beyond, considering how changes in mitochondrial metabolism or redox signaling can convert this interaction into a pathological event.

Impairments of cerebral blood flow microcirculation in rats brought on by cardiac cessation and respiratory arrest.

The impairments of cerebral blood flow microcirculation brought on by cardiac and respiratory arrest were assessed with multi-modal diagnostic facilities, utilising laser speckle contrast imaging, fluorescence spectroscopy and diffuse reflectance spectroscopy. The results of laser speckle contrast imaging show a notable reduction of cerebral blood flow in small and medium size vessels during a few minutes of respiratory arrest, while the same effect was observed in large sinuses and their branches during the circulatory cessation. Concurrently, the redox ratio assessed with fluorescence spectroscopy indicates progressing hypoxia, NADH accumulation and increase of FAD consumption. The results of diffuse reflectance spectra measurements display a more rapid grow of the perfusion of deoxygenated blood in case of circulatory impairment. In addition, consequent histopathological analysis performed by using new tissue staining procedure developed in-house. It shows notably higher reduction of size of the neurons due to their wrinkling within brain tissues influenced by circulation impair. Whereas, the brain tissues altered with the respiratory arrest demonstrate focal perivascular oedema and mild hypoxic changes of neuronal morphology. Thus, the study suggests that consequences of a cessation of cerebral blood flow become more dramatic and dangerous compare to respiratory arrest.

Polarization and depolarization metrics as optical markers in support to histopathology of ex vivo colon tissue.

Tissue polarimetry holds great promise to improve the effectiveness of conventional cancer diagnostics and staging, being a fast, minimally invasive, and low-cost optical technique. We introduce an enhanced diagnostic method for ex vivo colon specimens assessment by utilizing Stokes and Mueller matrix polarimetry. The proposed method makes use of experimental Mueller matrices, measured from healthy and tumor zones of a colon specimen, as input data for post-processing algorithms that include physical realisability filtering, symmetric decomposition and estimation of various polarization and depolarization metrics for colon specimen diagnostics. We validated our results with the gold standard histological diagnostics provided by pathologists. It was found that the Stokes-Mueller matrix polarimetry, combined with the appropriate filtering, decomposition algorithms and polarization/depolarization metrics calculations provides relevant optical markers of the colon tissue pathological conditions (healthy versus cancer), as confirmed by histopathology analysis. This approach potentially provides physicians with valuable and complementary information that holds promises in helping with the diagnostics of colon tissue specimens.

Assessment of Mitochondrial Membrane Potential and NADH Redox State in Acute Brain Slices.

Brain is one of the most energy-demanding organs. Energy in the form of ATP is produced in brain cells predominantly in oxidative phosphorylation coupled to mitochondrial respiration. Any alteration of the mitochondrial metabolism or prolonged ischemic or anoxic conditions can lead to serious neurological conditions, including neurodegenerative disorders. Assessment of mitochondrial metabolism is important for understanding physiological and pathological processes in the brain. Bioenergetics in central nervous system is dependent on multiple parameters including neuron-glia interactions and considering this, in vivo or ex vivo, the measurements of mitochondrial metabolism should also be complimenting the experiments on isolated mitochondria or cell cultures. To assess the mitochondrial function, there are several key bioenergetic parameters which indicate mitochondrial health. One of the major characteristics of mitochondria is the mitochondrial membrane potential (ΔΨm) which is used as a proton motive force for ATP production and generated by activity of the electron transport chain. Major donor of electrons for the mitochondrial respiratory chain is NADH. Here we demonstrate how to measure mitochondrial NADH/NAD(P)H autofluorescence and ΔΨm in acute brain slices in a time-dependent manner and provide information for the identification of NADH redox index, mitochondrial NADH pool, and the rate of NADH production in the Krebs cycle. Additionally, non-mitochondrial NADH/NADPH autofluorescence can signify the level of activity of the pentose phosphate pathway.

Spatial heterogeneity of cutaneous blood flow respiratory-related oscillations quantified via laser speckle contrast imaging.

LSCI technique provides experimental data which can be considered in the context of spatial blood flow coherency. Analysis of vascular tone oscillations gives additional information to ensure a better understanding of the mechanisms affecting microvascular physiology. The oscillations with different frequencies are due to different physiological mechanisms. The reasons for the generation of peripheral blood flow oscillations in the 0.14-0.6 Hz frequency band are as follows: cardio-respiratory interactions, pressure variations in the venous part of the circulatory system, and the effect of the sympathetic nervous system on the vascular tone. Earlier, we described the spatial heterogeneity of around 0.3 Hz oscillations and this motivated us to continue the research to find the conditions for the occurrence of spatial phase synchronization. For this purpose, a number of physiological tests (controlled respiration, breath holder, and venous occlusion tests) which influence the blood flow oscillations of 0.14-0.6 Hz were considered, an appropriate measurement system and the required data processing algorithms were developed. At spontaneous respiration, the oscillations with frequencies around 0.3 Hz were stochastic, whereas all the performed tests induced an increase in spatial coherence. The protocols and methods proposed here can help to clarify whether the heterogeneity of respiratory-related blood flow oscillations exists on the skin surface.

Skin Complications of Diabetes Mellitus Revealed by Polarized Hyperspectral Imaging and Machine Learning.

Aging and diabetes lead to protein glycation and cause dysfunction of collagen-containing tissues. The accompanying structural and functional changes of collagen significantly contribute to the development of various pathological malformations affecting the skin, blood vessels, and nerves, causing a number of complications, increasing disability risks and threat to life. In fact, no methods of non-invasive assessment of glycation and associated metabolic processes in biotissues or prediction of possible skin complications, e.g., ulcers, currently exist for endocrinologists and clinical diagnosis. In this publication, utilizing emerging photonics-based technology, innovative solutions in machine learning, and definitive physiological characteristics, we introduce a diagnostic approach capable of evaluating the skin complications of diabetes mellitus at the very earlier stage. The results of the feasibility studies, as well as the actual tests on patients with diabetes and healthy volunteers, clearly show the ability of the approach to differentiate diabetic and control groups. Furthermore, the developed in-house polarization-based hyperspectral imaging technique accomplished with the implementation of the artificial neural network provides new horizons in the study and diagnosis of age-related diseases.

Optical Diagnostics of the Maxillary Sinuses by Digital Diaphanoscopy Technology.

The work is devoted to the development of a scientific and technical basis for instrument implementation of a digital diaphanoscopy technology for the diagnosis of maxillary sinus inflammatory diseases taking into account the anatomical features of patients (differences in skin structure, skull bone thickness, and sinus size), the optical properties of exercised tissues, and the age and gender characteristics of patients. The technology is based on visualization and analysis of scattering patterns of low-intensity radiation as it passes through the maxillary sinuses. The article presents the experimental data obtained using the digital diaphanoscopy method and the results of numerical simulation of the optical radiation passage through the study area. The experimental setup has been modernized through the installation of a a device for controlling the LED applicator brightness. The approach proposed may have considerable promise for creating diagnostic criteria for various pathological changes and can be used to assess the differences in the optical and anatomical features of males and females.

Wearable Laser Doppler Flowmetry Sensor: A Feasibility Study with Smoker and Non-Smoker Volunteers.

Novel, non-invasive wearable laser Doppler flowmetry (LDF) devices measure real-time blood circulation of the left middle fingertip and the topside of the wrist of the left hand. The LDF signals are simultaneously recorded for fingertip and wrist. The amplitude of blood flow signals and wavelet analysis of the signal are used for the analysis of blood perfusion parameters. The aim of this pilot study is to validate the accuracy of blood circulation measurements recorded by one such non-invasive wearable LDF device for healthy young non-smokers and smokers. This study reveals a higher level of blood perfusion in the non-smoker group compared to the smoker group and vice-versa for the variation of pulse frequency. This result can be useful to assess the sensitivity of the wearable LDF sensor in determining the effect of nicotine for smokers as compared to non-smokers and also the blood microcirculation in smokers with different pathologies.

Machine Learning Aided Photonic Diagnostic System for Minimally Invasive Optically Guided Surgery in the Hepatoduodenal Area.

Abdominal cancer is a widely prevalent group of tumours with a high level of mortality if diagnosed at a late stage. Although the cancer death rates have in general declined over the past few decades, the mortality from tumours in the hepatoduodenal area has significantly increased in recent years. The broader use of minimal access surgery (MAS) for diagnostics and treatment can significantly improve the survival rate and quality of life of patients after surgery. This work aims to develop and characterise an appropriate technical implementation for tissue endogenous fluorescence (TEF) and assess the efficiency of machine learning methods for the real-time diagnosis of tumours in the hepatoduodenal area. In this paper, we present the results of the machine learning approach applied to the optically guided MAS. We have elaborated tissue fluorescence approach with a fibre-optic probe to record the TEF and blood perfusion parameters during MAS in patients with cancers in the hepatoduodenal area. The measurements from the laser Doppler flowmetry (LDF) channel were used as a sensor of the tissue vitality to reduce variability in TEF data. Also, we evaluated how the blood perfusion oscillations are changed in the tumour tissue. The evaluated amplitudes of the cardiac (0.6-1.6 Hz) and respiratory (0.2-0.6 Hz) oscillations was significantly higher in intact tissues (p < 0.001) compared to the cancerous ones, while the myogenic (0.2-0.06 Hz) oscillation did not demonstrate any statistically significant difference. Our results demonstrate that a fibre-optic TEF probe accompanied with ML algorithms such as k-Nearest Neighbours or AdaBoost is highly promising for the real-time in situ differentiation between cancerous and healthy tissues by detecting the information about the tissue type that is encoded in the fluorescence spectrum. Also, we show that the detection can be supplemented and enhanced by parallel collection and classification of blood perfusion oscillations.