Ilya E. Rafailov

Individual variability analysis of fluorescence parameters measured in skin with different levels of nutritive blood flow

Fluorescence spectroscopy has recently become more common in clinical medicine. However, there are still many unresolved issues related to the methodology and implementation of instruments with this technology. In this study, we aimed to assess individual variability of fluorescence parameters of endogenous markers (NADH, FAD, etc.) measured by fluorescent spectroscopy (FS) in situ and to analyse the factors that lead to a significant scatter of results. Most studied fluorophores have an acceptable scatter of values (mostly up to 30%) for diagnostic purposes. Here we provide evidence that the level of blood volume in tissue impacts FS data with a significant inverse correlation. The distribution function of the fluorescence intensity and the fluorescent contrast coefficient values are a function of the normal distribution for most of the studied fluorophores and the redox ratio. The effects of various physiological (different content of skin melanin) and technical (characteristics of optical filters) factors on the measurement results were additionally studied. The data on the variability of the measurement results in FS should be considered when interpreting the diagnostic parameters, as well as when developing new algorithms for data processing and FS devices.

Computational model of bladder tissue based on its measured optical properties

Abstract. Urinary bladder diseases are a common problem throughout the world and often difficult to accurately diagnose. Furthermore, they pose a heavy financial burden on health services. Urinary bladder tissue from male pigs was spectrophotometrically measured and the resulting data used to calculate the absorption, transmission, and reflectance parameters, along with the derived coefficients of scattering and absorption. These were employed to create a “generic” computational bladder model based on optical properties, simulating the propagation of photons through the tissue at different wavelengths. Using the Monte-Carlo method and fluorescence spectra of UV and blue excited wavelength, diagnostically important biomarkers were modeled. Additionally, the multifunctional noninvasive diagnostics system “LAKK-M” was used to gather fluorescence data to further provide essential comparisons. The ultimate goal of the study was to successfully simulate the effects of varying excited radiation wavelengths on bladder tissue to determine the effectiveness of photonics diagnostic devices. With increased accuracy, this model could be used to reliably aid in differentiating healthy and pathological tissues within the bladder and potentially other hollow organs.

Multimodal optical measurement for study of lower limb tissue viability in patients with diabetes mellitus.

According to the International Diabetes Federation, the challenge of early stage diagnosis and treatment effectiveness monitoring in diabetes is currently one of the highest priorities in modern healthcare. The potential of combined measurements of skin fluorescence and blood perfusion by the laser Doppler flowmetry method in diagnostics of low limb diabetes complications was evaluated. Using Monte Carlo probabilistic modeling, the diagnostic volume and depth of the diagnosis were evaluated. The experimental study involved 76 patients with type 2 diabetes mellitus. These patients were divided into two groups depending on the degree of complications. The control group consisted of 48 healthy volunteers. The local thermal stimulation was selected as a stimulus on the blood microcirculation system. The experimental studies have shown that diabetic patients have elevated values of normalized fluorescence amplitudes, as well as a lower perfusion response to local heating. In the group of people with diabetes with trophic ulcers, these parameters also significantly differ from the control and diabetes only groups. Thus, the intensity of skin fluorescence and level of tissue blood perfusion can act as markers for various degrees of complications from the beginning of diabetes to the formation of trophic ulcers.

The development of attenuation compensation models of fluorescence spectroscopy signals

This study examines the effect of blood absorption on the endogenous fluorescence signal intensity of biological tissues. Experimental studies were conducted to identify these effects. To register the fluorescence intensity, the fluorescence spectroscopy method was employed. The intensity of the blood flow was measured by laser Doppler flowmetry. We proposed one possible implementation of the Monte Carlo method for the theoretical analysis of the effect of blood on the fluorescence signals. The simulation is constructed as a four-layer skin optical model based on the known optical parameters of the skin with different levels of blood supply. With the help of the simulation, we demonstrate how the level of blood supply can affect the appearance of the fluorescence spectra. In addition, to describe the properties of biological tissue, which may affect the fluorescence spectra, we turned to the method of diffuse reflectance spectroscopy (DRS). Using the spectral data provided by the DRS, the tissue attenuation effect can be extracted and used to correct the fluorescence spectra.

A novel excitation-emission wavelength model to facilitate the diagnosis of urinary bladder diseases

Diseases of urinary bladder are a common healthcare problem world over. Diagnostic precision and predicting response to treatment are major issues. This study aims to create an optical cross-sectionional model of a bladder, capable of visually representing the passage of photons through the tissue layers. The absorption, transmission and reflectance data, along with the derived transmission coefficients (of scattering and absorption) were obtained from literature analysis and were used in the creation of a “generic” cross-section optical property model simulating the passage of thousands of photons through the tissue at different wavelengths. Fluorescence spectra of diagnostically relevant biomarkers excited by the UV and blue wavelengths were modelled on the basis of the Monte-Carlo method. Further to this, fluorescence data gathered by the “LAKK-M” system from pig bladders was applied to the model for a specific representation of the photon passage through the tissues. The ultimate goal of this study is to employ this model to simulate the effects of different laser wavelength and energy inputs to bladder tissue and to determine the effectiveness of potential photonics based devices for the diagnosis of bladder pathologies. The model will aid in observing differences between healthy and pathological bladder tissues registered by photonics based devices.

Detection of angiospastic disorders in the microcirculatory bed using laser diagnostics technologies

The evaluation of the microcirculatory bed functional state and the identification of angiospastic disorders with related complications, when the pathological changes are reversible, have an important role in medical practice. The aim of this study was to evaluate the possibility of using optical noninvasive methods and the cold pressor test to solve this problem. A total of 33 patients with rheumatological diseases and 32 healthy volunteers were included in the study. Laser Doppler flowmetry, tissue reflectance oximetry and pulse oximetry were used as optical noninvasive methods. The parameters were recorded before, immediately after and 20(Formula presented.)min after the cold pressor test. Based on the measured parameters, the complex parameters of the microcirculatory bed were calculated. A detailed statistical analysis of the parameter changes for each individual in the two groups displayed diverse microcirculatory bed parameter responses upon cold exposure, with differing recovery of parameters after CPT. New diagnostic criteria were proposed for the identification of angiospastic disorders. According to the proposed criteria, 27 people of the volunteers group were confirmed to not display any disorders. In the patient group, however, 18 people were observed to have a relatively normal functional state of the microcirculatory bed, while 15 people were observed to have a possible tendency to angiospasm. To highlight the differences between a relatively normal state and presence of angiospastic disorders, statistical analysis of experimental data was carried out, which revealed significant differences. Further analysis of data with angiospastic disorders identified a relationship between their diagnoses and the results of laboratory studies. Thus, the evaluation of combined noninvasive optical diagnostic method use, the cold pressor test and proposed diagnostic criteria showed a positive result. This approach can be used to detect the presence of possible angiospastic disorders and related complications, as well as microcirculatory bed disorders against the background of other diseases.

The blood perfusion and NADH/FAD content combined analysis in patients with diabetes foot

Skin blood microcirculation and the metabolism activity of tissue were examined on the patients with type 2 diabetes. Laser Doppler flowmetry (LDF) with 1064 nm laser light source and fluorescence spectroscopy (FS) with excitation light of 365 nm and 450 nm have been used to monitor the blood perfusion and the content of coenzymes NADH and FAD. Concluding, the proposed combined LDF and tissue FS approach allows to identify the significant violations in the blood microcirculation and metabolic activity for type 2 diabetes patients.

Laser Doppler flowmetry in blood and lymph monitoring, technical aspects and analysis

The aim of this work was to study the possibilities of the laser Doppler flowmetry method for the joint study of microhaemo- and lymph circulation of human skin. Conducting a series of experimental studies allowed to trace the relationship of recorded signals of microcirculation of blood flow and lymph flow, as well as to study their oscillation nature by using wavelet analysis.

A pilot study using laser-based technique for non-invasive diagnostics of hypertensive conditions in mice

Endothelial dysfunction is directly linked to preeclampsia, a maternal hypertensive condition that is life threating for both the mother and the baby. Epidemiological studies show that women with a history of pre-eclampsia have an elevated risk for cardiovascular disease. Here we report a new non-invasive diagnostic test for preeclampsia in mice that allows us to non-invasively assess the condition of the animals during the experiment and treatment in established models of preeclampsia. A laser-based multifunctional diagnostics system (LAKK-M) was chosen to carry out non-invasive analysis of multiple parameters. The device was used to simultaneously record the microcirculatory blood flow and oxygen saturation, as well as fluorescence levels of endogenous fluorophores. Preliminary experiments were conducted on adenoviral (Ad-)- mediated overexpression of sFlt-1 (Ad-sFlt-1) to mimic preeclampsialike symptoms in mice. The recorded data displayed the ability of the LAKK-M diagnostics device to detect significant differences in perfusion measurements between the control and Ad-sFlt-1 treatment. Preliminary results provide a potential avenue to employ these diagnostics technology to monitor and aid in maintaining control of live animal conditions throughout the experiment and treatment.

Fibre-optic probe for fluorescence diagnostics with blood influence compensation

To minimise the influence of blood content on the fluorescence measurements in vivo, a fibre optical probe combining fluorescence and diffuse reflectance measurements was developed. For the inverse solution of the blood content recovery, a set of neural networks trained by the Monte Carlo generated learning set was used. An approach of fluorescence measurements triggered by simultaneous real-time measurements of blood content in living tissue during moderate changes in contact pressure of the optic probe is proposed. The method allows one to decrease the necessary pressure on the probe as well as increase the repeatability of the measurements. The developed approach was verified in a series of experiments on volunteers with fluorescence excitation at 365 nm and 450 nm. The proposed technology is of particular interest in the development of new fluorescence-based optical biopsy systems.