This PDF file contains the front matter associated with SPIE Proceedings Volume 10685, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

Malignant gliomas are a devastating brain tumor disease with very poor prognosis. Stereotactic biopsy sampling is routinely used in larger neurosurgical centers to confirm the diagnosis of a suspected brain tumor. This procedure is associated with risk of blood vessel rupture as well as false-negative results. Recent investigations suggest a potential of light-based techniques to improve both therapy and diagnosis of GBM. Optical guidance can be utilized to improve the biopsy sampling procedure in terms of safety, reliability, and efficacy. Recording of optical signals (transmission, remission, fluorescence) can be potentially integrated into a biopsy needle for providing optical detection of tumor tissue and blood vessel recognition during the biopsy sampling. Optical signals can also be used for monitoring purposes during photodynamic therapy. Here, fluorescence signals recorded before the treatment indicate the presence and accumulation level of photosensitizer, while photobleaching of the photosensitizer fluorescence during the treatment can be used as a measure of the effectiveness of the therapy. Finally, transmitted light can reveal problematic tissue-optical conditions as well as changes of the optical properties of the treated tissue, which may be relevant with regard to treatment prognosis and strategy. Different optical concepts for interstitial PDT monitoring and optical tissue property assessment are presented.

The majority of denovo cancers are today being diagnosed in low and middle-income countries, which often lack resources and a range of therapeutic options. Minimally invasive therapies such as Photodynamic Therapy (PDT) and photobiomodulation (PBM) could become treatment options, albeit widespread acceptance is hindered by multiple factors ranging from training of surgeons in optical therapeutic techniques, lack of easily usable treatment optimizing tools and prediction of the anticipated treatment outcome. Based on the publicly available FullMonte software in combination with other open source image processing tools, a work plan is proposed that allows for personalized treatment planning. Starting with, generating 3D in silico models, execution of the Monte Carlo simulation and presentation of the 3D fluence rate distribution a treatment procedure is presented. Calculation of the forward solution of photon transport in biological tissues is executed in less than a minute for 3D models comprising 106 tetrahedral elements. The ability of the program to find optimal source placements was demonstrated for in silico brain tumour models for solid tumours. In hollow organs the impact of non-isotropic cavities is demonstrated on bladder cancer patient data. For photodynamic therapy treatment optimization, the process considers the selective uptake ratio of the photosensitizer between the target, host tissues and organs at risk and establish PDT sensitivities of these tissues based on the photodynamic threshold values. Tumours are assigned only a minimum required dose, whereas host and organs at risk a maximum permissible dose. For PBM the target and the host tissue are assigned minimum and maximum permissible dose due to the well documented biphasic response effect in PBM. For PDT sources of errors are uncertainties in the contouring whereas for PBM the depth of the actual target in the tissue is unknown and need often to be estimated based on body mass Index, and other morphometric parameters. Both photo therapeutic applications suffer from unknown tissue optical properties. Hence, the proposed workflow includes a perturbation of the planning tissue optical properties, uncertainties in the photon source placement and contouring errors, to validate the invariance of the attained solution against these unknowns. This requires also the need to determine the patients actual tissue optical properties at the onset of therapy, which in turn can only be achieved when the appropriate placement of invasive or diffuse reflective sensors is provided for. Hence, the planning process needs to include also identification of the most responsive positions for these sensors in the planning volume.

In photodynamic therapy (PDT) of tumors, targeted therapy is one of the most successful directions. The goal of the present work was to study the formation of new potential photosensitizers, based on transferrin (Tf) and cationic porphyrins, for targeted binding with transferrin receptors of cancer cells. We studied non-covalent binding of three cationic porphyrins 1) meso-tetra [4-N-(2'-oxyethtyl) pyridyl] porphyrin (TOEt4PyP) 2) Zn-TOEt4PyP and 3) Zn-mesotetra [4-N-butyl pyridyl] porphyrin (Zn-TBut4PyP) with human transferrin by absorption and fluorescent spectroscopy as well as by gel filtration methods. It was shown that the investigated porphyrins and metalloporphyrins bind stably enough to the protein molecule. It was found that the porphyrins having Zn ion in porphyrin core as well as the peripheral OH - groups are linked better to the transferrin molecules. It can be apparently explained by Zn coordination with transferrin amino acids and the formation of the hydrogen bonds between OH - groups of the porphyrin and transferrin amino acids. It was shown that, for the transferrin-porphyrin complexes, singlet oxygen luminescence is significantly decreased due to the presence of transferrin amino acids which are efficient quenchers of singlet oxygen.

The paper presents results on the response of living HeLa cells in vitro to low-dose photodynamic treatment with Radachlorin photosensitizer. Quantitative monitoring of variations of optical and morphological parameters of cells was performed by means of digital holographic microscopy and assisted with observations in confocal fluorescent microscope. The statistical analysis of the results obtained demonstrated significant morphological changes of cells along with invariable dry mass. The AO/EB standard test validated cell membrane integrity and demonstrated cells rounding and membrane blebbing. These data allow us to assume apoptosis as a major pathway of cell death activated in our experimental conditions.

The assessment of materials viscoelastic properties often represents a means of diagnosis or characterization of biological tissues and biomaterials. In this paper, we introduce a new optical method for the evaluation of dynamical properties of viscoelastic media. The approach is based on time-resolved spatial speckle imaging, using a continuous wave CW illumination and a standard CCD detector. We demonstrate that an estimation of viscoelastic properties is possible, by analyzing intensity and contrast profiles of scattering spot images acquired over multiple exposure times. The accuracy of this approach is evaluated using simulated tissue mimicking media having well known optical and dynamical properties.

Vascular activity is necessary to provide suitable energy supply for cellular activity in the brain. Obesity, has become an important health and social issue worldwide. Yet, very little is known regarding morphological and functional vascular changes in the brain in obese patients. The purpose of our study is to evaluate the influence of this pathology on blood flow, vasodilation and vasoconstriction at rest and during sensory stimulation, in normal and obese mice. In order to obtain dynamic and quantitative maps of vascular activity over wide field of cortical tissues in anesthetized mice brain, we have developed a multi-exposure speckle imaging (MESI) system. MESI relies on the sequential recording of speckle images of the brain tissues illuminated with coherent light for increasing durations. For each of these multi exposure images the local speckle contrast is derived. This contrast is assumed to be related to the velocity of scatterers (red blood cells). The acquisition of speckle contrast for different expositions time allows discriminating the contribution of static and moving scatterers to the speckle pattern. Therefore, it allows mapping the blood flow changes over large cortical areas. Blood flow response to sensory activation was studied by imaging the olfactory bulb during olfactory stimulation trials. Data obtained in wild -type and high fat diet obesity model mice are presented showing a different hemodynamic response to olfactory stimulation.

Laser speckle contrast imaging (LSCI) is a simple and quite powerful method for visualization of flow, microcirculation and perfusion. In current study the speckle contrast variations towards breaking ergodicity conditions are considered with a final aim of envision a practical approach allowing real-time imaging of variations in dynamic properties of complex fluids with an opportunity of quantitative interpretation of the obtained flowing map. As example of systems with static to dynamic transition, melting of Intralipid samples were studied. Also, investigation of influence of static layer thickness above the dynamic sample on the ergodicity condition has been studied.

Polarization imaging and Mueller polarimetry have shown great potentials as powerful tools for material science and biomedical studies. However, many polarization parameters are in uenced by the spatial orientation of the sample and experimental cofigurations. In this report, we introduce the rotation and mirror transformation theory of Mueller matrix, and propose a set of rotation invariant parameters and corresponding orientation predicting parameters. We analyze their physical meaning by studying the Mueller matrix of linear diattenuator, retarder and their combinations, conclude that the non-zero of some parameters can indicate the coexistence of difference anisotropy effects. Theories are verified with the Monte Carlo simulation of mixing aligned cylinder scatterers. It is shown that parameters from the Mueller matrix transformation theory are closely related to the symmetry properties of the sample, and have potential applications for fast analyzing different anisotropy contributions in the media.

Recently, a set of polarimetric indicators, the Indices of Polarimetric Purity (IPPs), were described in the literature. These indicators allow synthesize depolarization content of samples, and provide further analysis of depolarizers than other existing polarimetric indicators. We demonstrate the potential of the IPPs as a criterion to characterize and classify depolarizing samples. In particular, the method is firstly analyzed through a series of basic polarization experiments, and we prove how differences in the depolarizing capability of samples, concealed from the commonly used depolarization index P_Δ, are identified with the IPPs.

In the second part of this work, the method is experimentally highlighted by studying a rabbit leg ex-vivo sample. The obtained images of the ex-vivo sample illustrate how IPPs provide a significant enhancement in the image contrast of some biological tissues and, in some cases, present new information hidden in the usual polarimetric channels. Moreover, new physical interpretation of the sample can be derived from the IPPs which allow us to synthesize the depolarization behavior.

Finally, we also propose a pseudo-colored encoding of the IPPs information that provides an improved visualization of the samples. This last technique opens the possibility to highlight a specific tissue structure by properly adjusting the pseudo-colored formula.

Non-muscle invasive bladder cancer remains one of the costliest cancer to treat, and while a cystectomy will reduce a patient’s risk of developing metastatic disease it reduced the patient’s quality of life. While Photofrin mediate Photodynamic Therapy was approved already in 1993, poor control over the photon density and drug accumulation in of target tissue resulted in overdosing the bladder muscle layers, causing permanent volume shrinkage and incontinence. For an ongoing Phase Ib clinical trial, evaluating the safety of TLD1433 ([Ru(II)(4,4'-dimethyl-2,2'-bipyridine(dmb))2(2-(2',2'':5'',2'''-terthiophene)-imidazo[4,5-f][1,10]phenanthroline)]2+) a Ru(II) coordination complex, significant deviations from the previous studies are implemented. The photosensitizer is instilled, to reduce the sensitization of the muscle layer, 525 nm light is used to limit the light penetration into the bladder and the photon density is measured in each patient at up to 12 positions. Improved tumour selectivity is provided by this photosensitizer as it is block by the Urothelium from entering healthy tissue, whereas it enters tumour cells, supposing via the transferrin receptor, as demonstrated in in-vitro and in vivo studies. The Ru(II) coordination complex stains tissues where the urothelium is damaged very strongly in an orange-rust colour, visible under white light illumination. Preclinical in vivo studies showed the destruction of tumours up to 1 mm in depth following 1 hr of drug instillation, followed by 3 washes and the delivery of 90 Jcm-2 of 525nm light in the wistar rat Ay-27 tumour model. Histology showed very limited muscle damage and in general intact urothelium layers, with a moderate infiltration of macrophages. To achieve the prescribed target radiant exposure of 90 Jcm-2, independent of the bladder tissue diffuse reflectivity and shape, an optical dosimetry system was developed which can be deployed via a cystoscope. The optical dose monitoring device allows the treating physician to adjust the source position to achieve the target optical dose for the 12 sensor positions. The optical radiation was delivered via a 0.8 mm diameter spherical diffuser at up to 2.5 W power. The attainable photon density and the anticipated PDT dose are simulated for each patient using a photon propagation engine and the patient’s anatomical information. During these simulations, a range of tissue optical properties is simulated and compared to the initial photon density measurements to advise the physician further about the ability to achieve a homogenous illumination in each patient. The multiplication factor of the irradiance inside the bladder, calculated based on the spherical volume equivalent size of the bladder, the delivered power and measured irradiance, varied between patients (from 1.1 to 2.5) and also over the course of the treatment. The changes over the course of the treatment were predominantly due to light diffusing proteinaceous material floating in the bladder. Expanding prior work by the Rotterdam group on light propagation in the bladder we determined that the tissue albedo varied from 0.87 to 0.92 for 525 nm in this patient population. The estimated average effective attenuation coefficient for this population was approximately 1 mm adequate for the treatment of non-muscle invasive bladder cancer after transurethral resection of the large tumours. Monte Carlo modelling demonstrated that the fluence as function of depth into the tissue is determined by the exposure of bladder wall elements to the remaining bladder surface and can vary by factor of more than 2 even when assuming homogenous tissue optical properties across the bladder wall surface. These studies and analysis demonstrate the need for accurate light dosimetry in hollow organs when variations in the tissue albedo and the shape of the organ can influence the photodynamic dose significantly.

After irradiation with a blue LED light photocoagulator, a faster healing process is observed in superficial skin wounds. This device has been used in order to induce a thermal effect and haemostasis in superficial abrasions. Our previous in vivo study in rat and mouse models focused on the inflammatory phase within the healing process, showed a light-induced modulation, which leads to a shortened healing time and to a better recovery of the dermal tissue. Here we describe a new series of experiments that have been conducted producing two superficial abrasions on the shaved-back of mice, treating the one wound with the blue light and leaving the other one healing without any treatment. The healthy skin was used as a control. The animals were observed during healing and sacrificed at different and selected time points. Wound tissue samples have been harvested both from the treated and untreated areas and examined by histopathological and immunofluorescence analysis, SHG imaging, and confocal microscopy. The results of the study point out the interaction among different cells type and the collagen morphology restoration as obtained in different pathological mice models treated with blue LED light.

Novel products using nanosecond laser pulses such as LiDAR sensors gain more interest in the consumer industry. These products are in line with the coming trend and are assumed to gain economic importance in the future. The performance of such devices conforms to the maximum permissible exposures (MPE) of the laser safety standard. The accuracy of the standard can be improved by laser-induced damage measurements. Approaching this subject we present our own first ns-measurements of tissue damage in the mammalian eye and the interpretation of this data. The irradiation experiments were performed by means of a Q-switched, frequency-doubling Nd: YAG laser (532nm wavelength, 6 ns pulse). Freshly isolated bovine eyes were used as models for threshold determination of laser-induced thermo-mechanical damage. After removal of the neural retina the retinal pigment epithelium layer attached to the sclera was irradiated. Three spot diameters (150 µm 250 µm and 290 µm) were used. The evaluation of the data leads to the following ED₅₀ values: 3.55 µJ (150 µm), 6.50 µJ (250 µm) and 16.34µJ (290 µm). Based on the measurements, different options of data analysis are explained and used.

Retinoblastoma is a retinal cancerous disease that primarily affects young children. To date, preservation of the eye and its functionality is secondary to saving the child’s life. EpCAM+ Y79 retinoblastoma cells behave like cancer stem cells that are recognized as cells that are resistant to treatment. Additionally, reoccurrence of tumours is attributed to the persistence of cancer stem cells. An effective technique to treat retinoblastoma cancer cells is demonstrated using femtosecond laser pulses and EpCAM targeting gold nanorods (Au-NRs). Both fluorescence viability assay and MTS cellular metabolism assay confirm an astonishing cellular viability drop, to ~10%. It is shown that right after laser irradiation the cellular membrane ruptures. FESEM imaging shows that Au-NRs reach melting temperature after laser pulse exposure. The medium of the eye is transparent to NIR laser irradiation, making this treatment ideal for this type of cancer. This treatment methodology would also be an invaluable tool for treatment of chemotherapy-resistant and radiation-resistant cancers.

Food safety and quality is of worldwide concern and for meat the authentication of different species is a frequent issue with many implications including economic, religious, ethical, and health issues. Common analytical methods for meat species authentication are mostly labor-intensive, time-consuming and expensive. Optical techniques are a promising alternative enabling rapid and non-invasive in-situ analysis. This study extends our previous investigations to the analysis of frozen-thawed meat and meat juice using Shifted Excitation Raman Difference Spectroscopy (SERDS) applying two miniaturized SERDS probes operating at 783 nm (110 mW optical power, 0.5 nm spectral shift) and 671 nm (40 mW optical power, 0.7 nm spectral shift) that are fiber-optically coupled to compact spectrometers. Specimens comprise pork, beef, chicken and turkey and for each species 12 fresh meat slices were frozen at -18 °C for 7 days. After thawing each slice was measured at 15 different spots while for the meat juice 5 drops originating from each slice were analyzed recording 10 spectra with integration times of 10 seconds each. Partial least squares discriminant analysis models using 4 latent variables showed a clear distinction between individual species for meat (Sensitivity < 94 %, Specificity < 92 %) and meat juice (Sensitivity < 97 %, Specificity < 98 %). The classification is based on variations in myoglobin content and complex differences in protein Raman bands. The results underline the large potential of SERDS for rapid and non-invasive in-situ meat authentication paving the way for future applications at selected points along the process chain.

The state of internal human homeostasis, namely the function of the internal organs - the endocrine system, the digestive tract, the nervous, hematopoietic, cardiovascular and other systems, is closely related to the skin condition. Changes in the skin biochemistry are a reflection of the internal state of the human body. Therefore, the analysis of changes in the composition of human skin various layers is one of complex parts of therapeutic disciplines. In addition to the laboratory analysis methods used today, a variety of physical methods can be successfully used to study the component composition of the human skin. Methods of Raman Spectroscopy and autofluorescence analysis can detect changes in the component composition of the skin at the molecular level. In current study we used Raman spectroscopy and autofluorescence analysis in visible and NIR regions for the analysis of human skin spectral characteristics in the presence of various influencing factors including chronic kidney transplant dysfunction. The Raman and autofluorescence spectral characteristics of studied samples in NIR region were registered using the experimental setup, incorporated a high-resolution spectrometer with integrated cooled digital camera, a fiber-optic Raman probe and the laser module with central wavelength 785 nm. The autofluorescence human skin response in visible region was registered by portable diagnostic fluorimeter, which provide an excitation light source across the 350-400 nm range and measured light intensity within the 420-600 nm range. In this study we describe the design and results of the tests on volunteers of portable fluorescence meter based on two photodiodes. One channel of such fluorometer is used for measurement of autofluorescence intensity, another one - for intensity of elastically scattered radiation, which can be used as reference. The processing of experimental data was performed on the basis of regression analysis. We performed the comparative research of Raman experimental data and visible autofluorescence analysis results. We estimated correlations between Raman and autofluorescence signals and also find informative Raman bands that may be used as predictors of general condition of the body. These bands lie in 1170 – 1700 cm-1 region. We demonstrated the possibility to measure melanin and lipofuscin levels in the skin, as they are the hallmarks of skin aging; and demonstrated the possibility to measure a level of advanced glycation end products in the skin, advanced glycation end products as and lipofuscins are markers of general body condition. In addition, we have found informative spectral bands characterizing changes in the component composition of the skin in the presence of various influencing factors as kidney diseases.

Lipidomics is a vast field of intracellular pathways of lipids and their biochemical functions. In analogy to genomics and proteomics it contributes to the overall comprehension of system biology. The field elucidates the role of lipids as a subset of the major biological components. Within this family of molecules, often referred to as metabolic lipidome, lipid mediators (LMs) are currently under detailed investigations. Being part of lipid signaling events, which are unique in a sense that they are produced “on demand” at the site of action, LMs fulfill important roles in receptor and enzyme regulated processes. Furthermore, LMs along with phospholipids (PL) are known to have pro- and anti-tumoral properties, and cancer cells exhibit aberrant LM and PL profiles. Typical cells that produce a broad variety of LMs are monocytes and macrophages, which also use these chemical mediators to influence the communication between monocytes and macrophages with cancer cells. As lipidomics research involves the identification and quantification of the thousands of cellular lipid molecular species and their interactions with other lipids, proteins, and other metabolites, comparably fast analytical techniques that detect the overall lipid composition of individual cells are highly advantageous. Several types of analytical methodologies are applied for characterization of the lipidome of cells. By far most commonly used is mass spectrometry in combination with separation techniques that can provide a profile of the variety of lipids present, as well as their identification. Similar information can be obtained utilizing NMR spectroscopy. Meanwhile also well established for profiling biological samples is Raman spectroscopy. As Raman micro-spectroscopy can be used to image individual cells and depict subcellular components based on their spectroscopic fingerprints, it appears as an ideal label-free technique to investigate intracellular alterations noninvasively. minute spectral changes, due to compositional alterations can be reproducibly detected. In this context Raman micro-spectroscopy has for instance been applied to typing of bacteria or the differentiation between cancerous and normal cells. Raman spectroscopy can provide an OMIC-like view of the chemical status of individual cells and metabolism and has been suggested for lipidomic profiling.(1,2) The obtained data sets of were subjected to common statistical data evaluation, such as hierarchical cluster (HCA) and principal component analysis (PCA), in order to relate spectroscopic alterations to the compositional changes associated with the presence of a cancerous environment. Here we present first results obtained from M1 and M2 macrophages cocultured in vitro with cancer cells in order to evaluate the potential of Raman spectroscopy for lipid profiling. Acknowledgements: Financial support from the Carl Zeiss Foundation is highly acknowledged. References 1. Huang W, Spiers A. Consideration of Future Requirements for Raman Microbiology as an Examplar for the Ab Initio Development of Informatics Frameworks for Emergent OMICS Technologies OMICS: A Journal of Integrative Biology 2006;10:238-41. 2. Wu H, Volponi J, Oliver A, Parikh A, Simmons B, Singh S. In vivo lipidomics using single-cell Raman spectroscopy. Proc Natl Acad Sci USA 2011;108:3809-14.

We demonstrate resonance Raman spectroscopy in microfluidic channels for the analysis of whole blood. In particular, cell-free plasma layers are created in microfluidic whole blood flow by means of temporary hydrodynamic cell filter functionality. In-line confocal Raman spectroscopy is applied at the location of the created cell-free plasma layer and we detect free hemoglobin at diagnostic relevant hemolysis concentrations. Raman spectroscopy is a semi-quantitative method for chemical analysis. Due to the uniqueness of molecular vibrations, its selectivity is dependable. However, Raman scattering intensity is often too weak to be detected. This weakness can be overcome by resonance Raman spectroscopy where the laser excitation frequency is chosen corresponding to a dipole-allowed electronic transition of the molecule under study [1]. By employing resonance Raman spectroscopy we investigate bovine blood samples inside microfluidic channels in a micro-Raman setup, putting analytical emphasis on hemoglobin. Resonance Raman spectroscopy of hemoglobin was first demonstrated in 1972 [2] where aqueous solutions with concentrations of approximately 10-4 M were examined. Here we detect hemoglobin dissolved in bovine blood plasma inside 40μm deep PDMS channels. Most dominantly in our Raman spectra, the characteristic oscillatory mode of the central porphyrin ring structure of hemoglobin at 1375cm-1 appears. Despite the background in the Raman spectrum due to fluorescent emission from plasma proteins we are able to detect hemoglobin at concentrations from as low as 10-5 M and higher. The range of clinical relevance for hemolysis can be accurately resolved. The chemical analysis of liquid suspensions is of major interest to e.g. food industry, biotechnology and chemical industry. In this respect, it is desired to separate the suspending liquid from diluted particles, cells or beads [3]. A temporary particle or cell separation as proposed here is sufficient in order to analyze the suspending liquid in transit by micro-Raman spectroscopy. The microfluidic PDMS channels used in our whole blood experiments are 30-60μm wide and 40μm deep. In general, blood cells in flowing blood tend to migrate to the center of a microfluidic channel (Fåhræus–Lindqvist effect [4]), leaving a cell-free plasma region of 1-3μm at the channel walls. This cell-free plasma region can be locally expanded by the sudden enlargement of the microfluidic channel. In this way we create expanded semi-stagnant cell-free blood plasma regions of 5-20μm in width in close vicinity to whole blood flow. These regions are large enough to enable the application of localized confocal Raman spectroscopy exclusively in cell-free blood plasma. Hemolysis levels of whole bovine blood have been determined in this way. [1] M.D. Morris, D.J. Wallan, Anal. Chem., 51, 182–192 (1979) [2] T.C. Strekas and T.G. Spiro, Biochim. Biophys. Acta, 263, 830-833 (1972) [3] T. Kulrattanarak, R.G,M van der Sman, C.G. Schroën, R.M. Boom, Adv. Colloid Interface Sci, 142, 53-66 (2008) [4] R. Fahraeus, T. Lindqvist, The American Journal of Physiology, 96, 562–568 (1931)

We present a spectroscopic system and an optical fibre probe which enable the full exploitation of the temporal evolution and spectral information of a weak Raman signal against background fluorescence and intrinsic fibre Raman. The system consists of a single multimode fibre and a CMOS single-photon avalanche diode (SPAD) line sensor capable of resolving and histogramming the arrival times of photons for 256 pixels simultaneously, offering improved signal to background compared to a non-time resolved measurement modality. The capabilities of the system are tested for intrinsic Raman standards such as cyclohexane and for pH sensing with functionalised gold nanoshells exploiting surface enhanced Raman scattering (SERS). The nanoshells are functionalised with the pH responsive 4-mercaptobenzoic acid (MBA) enabling demonstration of wide range pH sensing with low excitation power (< 1 mW) and short acquisition times (10 s), achieving a measurement precision of ± 0.07 pH units.

The capability of brass as a real time substrate for surface enhanced Raman scattering applications is investigated. In this article, we showed that, using just the ultra-pure water as the electrolyte and the brass electrodes, ions extracted from the anode form nanoparticles on the anode surface in matter of minutes, and these nanoparticles are used for enhancing Raman signal intensity in real time. We observed enhancement factor of more than five orders of magnitude in Raman spectrum of Rhodamine B. We show that the nanoparticles formed on the brass anode surface are copper oxide nanoparticles and the enhancement of Raman signal intensity is due to these (copper oxide) nanoparticles. The zinc atoms do not affect the enhancement factor due to absence of zinc oxide nanoparticles on the anode surface. We present number of reasons to support this view in detail.

Recently, a new method called image reconstruction by integrating exchangeable single-molecule localization (IRIS) was developed (Kiuchi et al., Nat. Methods 2015, 12, 743-746). IRIS ensures high-density labeling for super-resolution microscopy but can be applied for fixed cells only. Here, we extended the IRIS conception to live cell imaging. We applied Lifeact and MAP4 transiently binding to microfilaments and microtubules, respectively. Green-to-red photoconvertible fluorescent protein Dendra2 was chosen as an efficient tag for single-molecule localization microscopy. Live-cell single-molecule localization imaging with Dendra2-Lifeact and Dendra2-MAP4 was performed. As a result, super-resolved images of actin and tubulin in dynamics in living cells were reconstructed. Importantly, Dendra2-Lifeact provided a higher number of individual localizations and denser labeling of microfilaments in comparison with commonly used Dendra2-actin.

Biosensors with high sensitivity are sought for a range of diagnostic applications, including detection of biomarkers at ultralow levels detection of disease risk, diagnosis or prognosis and as research tools to investigate questions of clinical relevance, e.g. profiling protein expression in single cell assays. Typical detection limits of bioassays in the market are in the nanomolar concentrations,1 while the emerging demands require sensitivity to be better by at least three orders of magnitude, viz. pico to the femtomolar regime. Achieving such high sensitivity is a significant challenge, where nanoplasmonic sensing considerable high promise. Nanoplasmonic sensing relies on giant electromagnetic field enhancements arising at the immediate vicinity of metal nanostructures upon excitation of localized surface plasmon polaritons by light irradiation at a suitable wavelength.2 For noble metals such as gold or silver EM field enhancements as high as 109-1012 can be realized in spatially confined volumes, defined by nanoscale gaps or curvatures, typically in the sub-10nm regime. Molecules that come in close vicinity to these regions of high EM enhancements (also known as hot-spots) can be detected via their vibrational Raman (surface-enhanced Raman scattering, or SERS) or fluorescence (metal-enhanced Fluorescence, or MEF) signals with detection limits potentially down to single molecule level. However, these confined volumes impose severe spatial constraints in accommodating biomolecular binding events, thus making it particularly challenging to take advantage of the high EM enhancements at the hot-spots. To this end, my thesis aims at rational design of nanoscale geometries that can enable co-localization of the reporter of biomolecular binding events with the plasmonic hot-spots to realize highly sensitive plasmonic biosensors based on surface-enhanced Raman spectroscopy. Scientific and Technical Challenges The key challenge for the thesis is to identify hot-spot configurations that present high EM enhancements which also allows sufficient space to accommodate biomolecular binding events. This requires both an ability to create and interrogate hot-spots, and controlling biomolecular events at the nanoscale. I. Engineered hot-spots a. High geometry control: The fabrication process to create hot-spots should deliver a high level of control over hot-spot geometries, with geometric attributes that are consistent across the sample and reproducible across batches, preferably within a tolerance of 10%. This is necessary to achieve high enhancement factors, with low signal intensity variations to facilitate rational design through modelling and simulations and to avoid false negatives in the eventual biosensor. b. High density of hot-spots spanning large area: This will facilitate higher signal levels, and also allow larger dynamic range for sensing. The larger area would enable use of probing tools/techniques with a macroscopic footprint (of several microns to square millimetres). Achieving this may impose a heavy penalty of cost or time using most commonly available nanofabrication tools that deliver high control (e.g. e-beam lithography, focused ion beam lithography) c. Amenable to rational design: Rational design requires systematic investigations requiring correlations between process parameters, nanostructure geometry, optical/spectroscopic property. This requires a geometry that can be readily modelled, with a minimal deviation of such model from experimentally realized geometries. d. The hot-spot geometry should take into account the length-scales of the biomolecular binding event for the bioassay in question. For a typical immunoassay, the binding event can measure up to 40 nm.3 e. The characterization of the geometry of hot-spot is not always straightforward. The solution has relied on building an understanding based on information acquired from multiple techniques, e.g. SEM, AFM, geometric model, optical simulations. II. Biomolecular binding events at confined spaces a. Attachment of biomolecules: Attempts to position biomolecules at hot-spots using typical strategies employed for patterning biomolecules would require selective surface functionalization at length scales of only a few nanometers. This imposes the severe challenge to preserving the integrity of the final functionalized surface. b. Understanding the nano-bio interface: Nanostructures on the surface are known to influence either favourably or adversely the interaction of biomolecules with surface.4 Such interaction can be tailored by controlling the environment (e.g. pH, the ionic strength of the medium, the zeta potential on the surface, etc.). It is a challenge to coordinate the fabrication to ensure that the plasmonic interface can be subjected to the biosensing experiments and the multiple changes to the biosensing environment without losing stability.

Raman spectroscopy has numerous applications in the field of biology. One such application is the simultaneously measurement of the concentration of multiple biochemical components in low volume aqueous mixtures, for example, a single drop of blood serum. Over twenty years ago, it was shown for the first time that it was possible to estimate the concentration of glucose, urea, and lactic acid in mixture by combining Raman Spectroscopy with Partial Least Squares Regression analysis. This was followed by numerous contributions in the literature designed to increase the number of components and reduce the limits of concentration that could be simultaneously measured using Raman spectroscopy, by developing various optical architectures to maximise the signal to noise ratio. The aim of this paper is to demonstrate the potential of a confocal Raman microscopy system for multicomponent analysis for the case of physiologically relevant mixtures of glucose, urea, and lactic acid.

A biosensor platform based on Bloch Surface Waves and operating in angular interrogation mode is applied to the detection of a clinical biomarker (HER2-neu/ERBB2) related to breast cancer initiation/progression. Preparing regions for specific recognition of different proteins as well as a reference on the biochip enables to correct the signal for nonspecific effects. Additionally, label-free analysis and surface wave enhanced fluorescence detection can be applied and compared directly on the platform. Cell lysates with high and low expression levels of ERBB2 are analyzed. Comparing the signals of such ERBB2 positive and negative samples estimates the limit of detection at 1.7 ng/mL. This is well below the threshold of 15 ng/mL set by the FDA for clinically useful ERBB2 detection in human serum, demonstrating that 1DPC-based biochips are attractive candidates for breast cancer detection/monitoring.

In this paper, we develop dielectric micro-optical sensors based on whispering gallery mode phenomenon (WGM) for monitoring and treating of drinking water environments through two phases. Some sort of chemical impurities could be toxic and carcinogenic to humans and animals. The biogeochemical reactions are governing the chance and the movement of these impurities in the drinking water environment. Based on that, the first phase in this paper will focus to measure and quantify the concentration of these impurities in the water medium. While the second phase will exploit the use of the light based on the same phenomena (WGM) to create water treatment and purification using a nano charged dielectric polymeric beads. In the current paper, a high-resolution micro-optical sensor concept is used to detect these chemical impurities. The sensing element is a silica microsphere acts as an optical resonator. The proposed technique aims to provide preliminary results demonstrating the practical success of these sensors for effective monitoring of chemical impurities concentrations and contaminants which can cause serious kidney damage and possibly death. The second phase is basically depend on the optogenetic approach which is a biological technique that involves the use of light to control cells in living tissue, typically neurons that have been genetically modified to express light-sensitive ion channels. In this approach, the beads will be coated with a photosensitive protein called channelrhodopsin. This protein is a subfamily of retinylidene proteins (rhodopsins) that function as light-gated ion channels. They serve as sensory photoreceptors in unicellular green algae, controlling phototoxic: movement in response to light. The nano coated beads then poled for +4hrs under 1MV/m. When these nano charged beads mixed with water that have high turbidity, the beads starts to attract the colloids in that water. Since, the beads are coated with a photosensitive protein so by using a specific wavelength of the light we can control the motion of the spheres inside the water. Using a pulse width modulation (PWM) algorithm to control the speed of switching on/off the light; so it becomes easy to control the nano beads. The higher duty cycles for the PWM the charged beads makes the colloids aggregate and come together in a very short time (< 5 min) compared to the typical flocculation approaches that needs (~55min). This approach is called an optical flocculation technique and it shows one order of magnitude enhancement in the flocculation time. Results indicate that the WGM based-sensors are sensitive enough to refractive index changes in the case of liquid media (water). Experiments were carried out to validate the analysis and to provide an assessment of this sensor concept. Also, Preliminary experiments were carried out to provide an assessment of this concept using more than one duty cycle to control the speed of the beads. Results shows that we can purify the drinking water in time less than 3 minutes under 80% duty cycle using this approach.

Myocardial infarction (MI) causes partial or complete necrosis of the heart muscle. It means that muscle cells are wiped, and the contractility of the heart decreases. Today, for the MI diagnosis is based on the ECG recording or specific biomarkers identifying in the patient's blood, the most specific of which are: creatine phosphokinase (CPK), CPK-MB, fatty acids binding protein (H-FABP), myoglobin troponin-I, and troponin-T.

Additionally to these biomarkers in the MI patients’ blood, there are many other products of metabolism in damaged muscles, which are excreted from the body human body, including through exhaled air. The results of MI patients’ exhaled air analysis using photoacoustic laser spectroscopy and data mining are presented.

A periodic monitoring of the adipose tissue functions due to interventions, such as calorie restriction and bariatric surgery, or pathophysiological processes, has an increasing relevance in clinical diagnostics. Diffuse Optical Spectroscopy (DOS) is a valuable non-invasive tool that can be used in that direction. In this work, we present a pilot study based on Time Domain Broadband Diffuse Optical Spectroscopy (TD DOS) to characterize in vivo the subcutaneous fat tissue in the abdominal region. A first of its kind, portable TD DOS instrumentation, already enrolled in clinical studies, was used. Three healthy male volunteers were considered. Three source-detector separation distances (1, 2, and 3 cm) were used over the broad wavelength range of 600-1100 nm. The analysis was performed using a method based on a heterogeneous model to account for the multi-layered nature of the subcutaneous adipose tissue, and to obtain the optical properties specific to this fat localization. Inter-subject variation of tissue composition data was observed.

Light is a powerful non-invasive tool that can be exploited to probe highly scattering media like biological tissues for different purposes, from the detection of brain activity to the characterization of cancer lesions. In the last decade, timedomain diffuse optics (TDDO) systems demonstrated improved sensitivity when using time-gated acquisition chains and short source-detector separations (ρ), both theoretically and experimentally. However, the sensitivity to localized absorption changes buried inside a diffusive medium strongly depends on many parameters such as: SDS, laser power, delay and width of the gating window, absorption and scattering properties of the medium, instrument response function (IRF) shape, etc. In particular, relevant effects due to slow tails in the IRF were noticed, with detrimental effects on performances. We present simulated experimental results based on the diffusion approximation of the Radiative Transfer Equation and the perturbation theory subjected to the Born approximation. To quantify the system sensitivity to deep (few cm) and localized absorption perturbations, we exploited contrast and contrast-to-noise ratio (CNR), which are internationally agreed on standardized figures of merit. The purpose of this study is to determine which parameters have the greatest impact on these figures of merit, thus also providing a range of best operative conditions. The study is composed by two main stages: the former is a comparison between simulations and measurements on tissue-mimicking phantom, while the latter is a broad simulation study in which all relevant parameters are tuned to determine optimal measurement conditions. This study essentially demonstrates that under the influence of the slow tails in the IRF, the use of a small SDS no longer corresponds to optimal contrast and CNR. This work sets the ground for future studies with next-generation of TDDO components, presently under development, providing useful hints on relevant features to which one should take care when designing TDDO components.

A diagnostic fluorimeter has been developed to estimate in vivo the content of the advanced glycation endproducts in the human skin using autofluorescence. It consists of two light-emitting diodes and implements a pulse current source for the LEDs. The LED with a peak wavelength of 365 nm is used as excitation source. Green LED with is used to obtain the information about the patient's skin phototype. The analog electronics of the fluorimeter includes two photodetector channels using silicon photodiodes. Pulse modulation of LED light made it possible to reduce the influence of sunlight and illumination on a digital signal at the output of a 10-bit ADC to a level of less than least significant bit in both channels. The digital part of the fluorimeter is based on the Arduino platform. The software controls the operating modes of the fluorimeter, provides a quantitative processing of the results, visualizes and saves the diagnostic data. Experimental studies have demonstrated the operability of the device and its noise immunity, the ability to identify agerelated changes in the skin. Nevertheless, a significant contribution to the statistical spread of the values of the diagnostic parameter is made by uncontrolled changes in the diagnosed area of the skin on the inner side of the forearm when it detaches and again touches the entrance window of the fluorimeter. The accepted normalization of the autofluorescence signal to the product of elastic skin scattering signals from the ultraviolet and green light-emitting diodes allowed to reduce the variability of the diagnostic parameter due to sunburn by a factor of 1.5.

Most surgeries are currently performed subjectively, with outcomes that are largely dependent on the experience of the surgeon. Providing objective information about tissue that need to be resected or avoided could reduce the variability in surgical outcomes. Spatial Frequency Domain Imaging (SFDI) is a novel diffuse optical imaging method that has the potential to identify tissue viability over a large field of view. In this method, a spatial sinusoidal pattern is projected onto the tissue to get the optical properties of the tissue at each pixel. More recently, Single Snapshot of Optical Properties (SSOP) hase been developed to provide such image feedback in real-time within the specific constraints of surgery. However, while SSOP has been shown to provide information about tissues at a single wavelength in real-time, during surgical applications, it is critical to obtain spectrally-resolved functional information that can be easily interpreted by the surgeons. Optical properties at multiple wavelengths are therefore needed to correlate the absorption and scattering coefficients with the tissue functional and structural information. In this work, we propose a novel method relying on spatio-temporal modulation of light to obtain multispectral optical properties in real time. A temporal-encoding method is used to distinguish different wavelengths by modulating each wavelength at a one particular chosen frequency. The temporally-modulated light is then used to project sinusoidal patterns onto the tissues for SSOP processing. The scene is then recorded with a fast camera to get multiple information in 3 dimensions: two spatial dimensions for SSOP and the third temporal dimension for wavelength. Discrete Fourier Transform (DFT) is used to separate the modulation frequencies pixels by pixels, and each 2D image obtained for every wavelength is processed using SSOP to obtain absorption and scattering coefficients. Finally, the optical properties at each wavelength are used to provide functional and structural information about tissues. We validated this proof of concept using 2 wavelengths (665 and 860nm) during phantom measurements and in vivo by obtaining real-time oxygenation videos. This work lays the foundation for the clinical translation of real-time quantitative multispectral imaging.

Intra-operative fluorescence-guided resection (FGR) enables maximum safe resection of glioma by providing real-time tumor contrast. In its most widely used form, FGR is mediated by the preferential overproduction of the fluorophore protoporphyrinIX (PpIX) in malignant tissue after an oral dose of its precursor 5-Aminolevulinic Acid (ALA)1. ALA-PpIX-FGR has been shown to significantly increase completeness of tumor resection. However, the subjective visual assessment and the variable intrinsic optical attenuation of tissue limit this technique to delineating only high-grade tumors that display strong fluorescence residing at the tissue surface. We have shown that wide-field quantitative assessment by extracting 2D maps of PpIX concentration in the tissue, [PpIX], significantly improves the accuracy in detecting diffuse tumors, thereby potentially extending FGR to patients with low-grade tumors. In this approach, hyperspectral fluorescence imaging is coupled to a custom-built spatial frequency domain imaging (SFDI) system. SFDI enables the recovery of tissue optical properties maps, μ_a and μ_s^'. These are used to correct the fluorescence images. The corrected hyperspectral fluorescence images are then spectrally unmixed to separate true PpIX fluorescence from that of its photoproducts and from autofluorescence. Quantitative fluorescence imaging was validated against the clinically used spectroscopic probe by comparing the recovered optical properties and [PpIX] in vivo of a rat brain tumor model. This quantitative approach was also applied to a near infrared fluorophore ZW-800 on tissue-simulating phantoms. ALA-PpIX-FGR, as it is currently implemented, is inaccessible to infiltrative residuals lying beyond the resection cavity because of the limited penetration depth of the blue excitation light used. This is problematic as these infiltrative tumors are the main cause of reccurence. Enhanced sub-surface tumor detection was shown feasible by exciting PpIX’s secondary absorption peak of 635 nm intra-operatively on patients with various intra-cranial pathologies. However, resolving strong fluorescence of a deep-seated tumor from weak fluorescence of a shallow tumor was not possible. That is because the detected fluorescence intensity is heavily dependent on fluorophore concentration, depth, and fluorophore distribution, while also being convolved with tissue turbidity. The aim of this work, therefore, is to extend quantitative ALA-PpIX-FGR to identify sub-surface tumors by resolving tumor depth from fluorophore concentration. This should assist the surgeon in making an informed decision as for whether to further resect or not. A new quantitative depth imaging method was developed by exploiting SFDI’s depth-encoding capabilities in fluorescence mode. The result is a series of spatially modulated fluorescence images, where the modulation amplitude decays with increasing spatial frequency at a rate dependent on fluorophore depth. After recovering depth, a diffusion-based fluorescent light transport model is applied to extract fluorophore concentration. The algorithm was validated using tissue-simulating phantoms and an ex vivo tissue model indicating that the maximum depth recovered is highly dependent on fluorophore concentration as well as on tissue turbidity. For the [PpIX] and optical property maps relevant for glioma tissue, our quantitative depth fluorescence technique can predict depths up to 9 mm ± 0.4 mm, while recovering [PpIX] with an accuracy of 15% for concentrations as low as 2.5 µg/ml.

Although laserosteotomes have become generally accepted devices in surgical applications, they still suffer from a lack of information about the type of tissue currently being ablated; as a result, critical structures of the body under or near the focal spot of the laser beam are prone to inadvertent ablation. The lack of information about the properties of the ablated tissue can be solved by connecting the laserosteotome to an optical detection setup which can differentiate various types of tissues, especially bone from connective soft tissues. This study examines the applicability of laser-induced breakdown spectroscopy (LIBS) as a potential technique to differentiate bone from surrounding soft tissue (fat and muscle). In this experiment, fresh porcine femur bone, muscle, and fat were used as hard and soft tissue samples. The beam of a nanosecond frequency–doubled Nd:YAG laser was used to ablate the tissue samples and generate the plasma. The plasma light emitted from the ablated spot, which corresponds to the recombination spectra of ionized atoms and molecules, was gathered with a collection optic (including a reflective light collector and a fiber optic) and sent to an Echelle spectrometer for resolving the atomic composition of the ablated sample. Afterwards, Discriminant Function Analysis (DFA) based on the ratio of the intensity of selected peak pairs was performed to classify three sample groups (bone, muscle, and fat). Lastly, the sensitivity, specificity, and accuracy of the proposed method were calculated. Sensitivity and specificity of 100 % and 99 % were achieved, respectively, to differentiate bone from surrounding soft tissue.

Skin capillary blood oxygen saturation is a clinically important diagnostic parameter, which provides valuable information for timely treatment of pathological conditions e.g. sepsis, hypoxemia or decompression illness. Hyperspectral imaging is non-invasive optical techniques with high clinical potential, however its use for skin blood oxygen saturation detection is still challenging, therefore in the present study, a method for in-vivo manipulation of skin oxygen saturation was developed, and reliability of the method evaluated by means of hyperspectral imaging in detection of oxygen saturation. In order to produce alterations of skin capillary blood parameters and oxygen saturation, the proximal phalanx of the right middle finger was occluded with a pneumatic cuff for 25 minutes. During the last minute of occlusion, the hyperspectral cubes (HIS) of both occluded and intact finger were captured, and capillary blood sample was collected for analysis with portable whole blood analyzer (REF). The group mean values for SaO2 in intact finger skin was HIS: 89.46%±8.79% versus REF: 95.13±1.46 % and in occluded finger HSI: 25.85% ±14.00%, versus REF: 22.73±9.09 % displaying a small difference between two independent techniques, which indicate the reliability of finger occlusion model.

Malignant melanoma is the most dangerous oncological skin disease and, as a rule, rapidly progresses, spreading metastases throughout the body. In this way, it is important to diagnose this as early as possible, on early stages. However, this process requires a physician who does not always have enough time and experience to analyze a suspicious neoplasm. In this research, we have developed a device for dermatoscopic skin examination. The own developed Dermatoscope is based on the Basler acA1920-25uc camera (RGB, 12 bit/px, 1920*1080) has a resolution of ~13 μm/px on the surface of the skin. The device body has been designed and printed on a 3D printer. Tissue lighting is carried out by various LEDs: • UV: 2 x LEUVA77V20RV00 (365 nm, 1 W, 9 nm half-width); • White: 4 x LEDs FM-5630WDS-460W-R80 (39 lm, 4000 K) (two polarized LEDs and two unpolarized ones); • Blue: 2 x CREE XREBLU-L1-0000-00K01 (30 lm, 465-485 nm); • Green: 2 x CREE XRCGRN-L1-0000-00N01 (52 lm, 520-535 nm); • Red: 2 x CREE XPCReD-L1-0000-00301 (46 lm, 620-630 nm). The device takes six pictures during approximately 6 sec with different light on user’s request. Development prototype is 3D printed with ABS plastic. Used lights are white light, white polarized light for deeper skin layers visualization, UV light for fluorescence analysis, three visible colored lights for skin visualization is red, green and blue wavelength ranges which could be used for oxyhemoglobin, deoxyhemoglobin and melanin chromophore mapping. Using linear polarizing filter allows us glares filtering. Polarizing filter has been used with white light and visible colored lights in the development dermatoscope prototype. It allows us to visualize deeper skin layers and get light absorption at three visible wavelengths without glares influence. The second polarizing filter has been placed on the lens, so all of the filters placed on light sources must be oriented in one direction. Scattered excitation wavelength light must be filtered while allowing longer wavelengths to pass to get skin autofluorescence images. A 430 nm long-pass filter has been installed on the lens for this purpose. This device is also equipped with software for automatic recognition of skin melanoma based on texture (Haar wavelet texture features, local binary patterns) and color analysis. In this paper, we present the results of analysis of 225 multispectral images of melanoma and other benign and malignant tumors of the skin in vivo. The dermatoscopy tool analyzes pictures of suspicious areas, highlighting contrast areas of the skin and compares with existing diagnosed cases, which speeds up the accuracy of diagnosis. Using our software, high accuracy (96% sensitivity and 89% specificity) for melanoma has been achieved, which means a principal opportunity of effective excluding similar formations that are not melanoma. Thus, the proposed dermatosocpy tool can be used for screening procedures.

Optogenetics utilizes light sensitive proteins to trigger biological functions by an external optical stimulus. It has been applied in numerous settings, including robotics, biotechnology, and several therapeutic approaches. The latter is of particular interest, as optogenetics provides the possibility to directly control cellular functions of excitable tissues, such as neurons and muscles, in a contact free manner by ion channels such as channelrhodopsin-2. In this context, optogenetics allows to create a human-implant interface, with an unprecedented performance. However, optogenetic therapeutic approaches are inhibited two conceptual constraints, limiting their clinical translation. (1) Precise and defined delivery of the stimulation light is restricted by scattering and absorption of the body’s tissue, limiting its resolution and thereby selectivity. (2) Optogenetics implies intrinsically a gene modification of the target tissue, raising issues concerning the effective in vivo delivery and the safety of the (viral) vectors applied. Biocompatible waveguides which in parallel can serve as cell encapsulation could provide a promising approach to circumvent these constraints. Encapsulation would ensure optical accessibility and encase the genetically modified cells. These constructs should guide and distribute the light as desired with minimal losses and they should also mimic the mechanical properties of the surrounding tissue to ensure compatibility and long-term stability. In this project, we present a study on poly(ethylene glycol) (PEGDA) based hydrogels as waveguides for optogenetic pacing of the heart. PEGDA is a non-biodegradable, biocompatible polymer with high transparency for visible wavelengths. Both optical and mechanical properties of hydrogels made from PEGDA derivatives are tuned to achieve compatibility with muscle tissue as well as optimal light guiding and distribution. The excitation light is coupled into the hydrogel with a biocompatible fiber. The design of the fiber-hydrogel contact must ensure efficient light coupling as well as mechanically stable binding of the fiber to the hydrogel construct. Properties of the hydrogel are mainly tuned by monomer length and concentration. Total internal reflection can be achieved by embedding a fiber-like hydrogel with a high refractive index into a second, low refractive index gel. Multi-component gels and different geometries are explored as additional ways to impact light distribution. After optimization, the hydrogel may be used to deliver light for the excitation of genetically altered cardiomyocytes for controlled contraction. This would pave the way for the development of a biohybrid, optogenetically driven pacemaker implant. On a long term perspective, hydrogel waveguides with embedded optogenetic excitable cells which functionally couple to the target tissue may serve as a general human-implant interface, with encased genetic modification and controlled, biocompatible light guiding to the target tissue.

In this paper, we study the effect of using magnetorheological polydimethylsiloxane (MR-PDMS) spheres as micro optical resonators. Recently, optical sensors based on whispering gallery mode (WGM) have drawn much attention due to the high optical quality factors they can exhibit with several applications in a variety of fields. The modes of such resonators are highly affected by changes in morphology of the cavity (for instance: shape, size or refractive index) caused by a change in the physical input we are aiming to measure. In the presence of a magnetic field, the embedded particles become polarized, exerting forces upon one another because of dipolar interactions. In our case, these forces lead to changes in the morphology of the resonator through mechanical deformation of the size, which in turn causes WGM shifts. Experiments were conducted on earthworms for the similarity of the nerve impulses flowing which mimic those of human body specifically the brain and ultra-small magnetic fields can then be detected. Moreover, the coupling of a MR cantilever beam to the optical resonator leads to additional induced forces for the same magnetic field input and thus increasing the resolution by 3 orders of magnitude. Results indicate that the proposed sensor can be used for magnetic field detection with high quality factor and resolution.

Terahertz (THz) pulsed spectroscopy is a convenient instrument for studying the THz dielectric response of healthy and abnormal tissue in a wide spectral range. One of the most promising applications of THz pulsed spectroscopy is associated with non-invasive, least-invasive and intraoperative medical diagnostics of malignancies in various localizations, including the skin, the breast, the colon, and the brain [1]. In our research, we developed a method for reconstructing the THz dielectric response of biotissues in vitro and in vivo using the THz pulsed spectroscopy [2–5]. We applied this method for studying healthy and pathological tissues of the skin and the brain. (i) We observed statistical differences between THz dielectric properties of ordinary and dysplastic nevi of the skin in vivo. This highlights an ability for non-invasive early diagnosis of dysplastic nevi and melanomas of the skin using the THz spectroscopy and imaging [3–5]. (ii) By studying the THz dielectric permittivity of non-melanoma skin cancers in vitro (i.e. basal cell carcinoma and squamous cell carcinoma), we justify an ability for discriminating malignant tissues from surrounding normal skin using preoperative and intraoperative THz imaging [6,7]. (iii) Finally, the results of measuring the THz dielectric response of gelatin-fixed malignancies of the brain in vitro allow us to analyze an ability for discriminating brain gliomas from surrounding normal tissues during the neurosurgery using the THz technologies. The observed results of THz measurements agrees well with the data of biotissues studying using other modern modalities of optical imaging, such as intraoperative exogenous fluorescence imaging and optical coherence tomography, as well as with the data of biotissue histology. These results highlight the prospective of THz spectroscopy, imaging and endoscopy use for non-invasive, least-invasive and intraoperative medical diagnosis of malignancies. [1] O.A. Smolyanskaya,·M.M. Nazarov,·O.P. Cherkasova,·J.-P. Guillet,·J.-L. Coutaz, A.A. Konovko, Y.V.Kistenev,·P. Mounaix, I.A. Ozheredov, V.L. Vaks, A. Yaroslavsky,·N.V. Chernomyrdin, K.I. Zaytsev, S.A. Kozlov,·J.-H. Son, V. Wallace,·A.P. Shkurinov, ·V.V. Tuchin, “Terahertz biophotonics as a tool for studies of dielectricand spectral properties of tissues and bioliquids relatedto water content,” Progress in Quantum Electronics (2017, Submitted). [2] IEEE Transactions on Terahertz Science and Technology 5(5), 817 (2015). [3] Applied Physics Letters 106(5), 053702 (2015) [4] European Journal of Cancer 51, S167 (2015). [5] Optics and Spectroscopy 119(3), 404 (2015). [6] Journal of Physics: Conference Series 486(1), 012014 (2014). [7] Journal of Physics: Conference Series 584(1), 012023 (2015).

Optical spectra acquired on skin depend greatly on skin content in chromophores especially on photons absorbers (melanin) and on fluorescent molecules such as collagen and elastin. Such skin content in chromophores varies from one person to another one even within the same phototype class. Therefore, optical spectra must be standardized in order to be as independent as possible of inter-individual variability. Such a standardization should help increase the diagnosis accuracy of optical spectroscopy. In this study, we aim at defining the anatomical site that would allow best standardization. Standardization is evaluated through the ability of spectroscopy to discriminate pairs of histological classes such as BCC, SCC, AK and normal skin. Bimodal spectroscopy is used combining AutoFluorescence (AF) and Diffuse Reflectance (DR). Three anatomical sites are compared for reference spectra acquisition: non-lesional (NL) skin sites (i.e. the same anatomical sites as for BCC, SCC or AK), inner hand and inner wrist. AF raw data show a high (40 %) inter-individual variability of reference spectra intensity acquired on any of the three anatomical sites. Standardization using reference spectra allow best discrimination of histological classes when reference spectra acquired on NL skin sites and on inner hand are used for standardization of AF spectroscopy and DR spectroscopy, respectively.

Medical fiber optics as a part of laboratory and clinical diagnostic tools, surgical instrumentation and endoscopy should satisfy modern biomedical requirements. The flexibility, small size, bio-compatibility and feasibility to use sterilisable or disposable parts allow to apply fiber optic probes in clinical environment both ex-vivo and in-vivo. To enable spectroscopic differentiation cancer and normal tissue, we developed and applied various single and combined fiber optic probes using key spectroscopy methods such as Raman scattering, Mid IR-absorption, Diffuse NIR-reflection, and fluorescence in order to compare them and select the best combination for malignant detection of tissue in clinical environment. All four spectroscopic methods have been tested on biopsies of health and malignant tissues (colon and kidney) and bioliquids (serum, plasma and urine) of patients before and after surgery. The tiny Raman probe with 1.5 mm diameter has been developed for experimental ex-vivo tests. Further multivariate data analysis of spectroscopic data, both individual techniques and their combinations has provided a reliable cancer recognition for colon and kidney biopsies. The best synergic gain was observed of combining Mid IR-absorption and fluorescence spectroscopy (98% Sensitivity vs 63% or 88% for fluorescence or Mid IR-absorption correspondingly). Based on obtained results, both techniques were implemented within the same fiber probe to provide a simultaneous measurement of exactly the same spot at the sample surface. An innovative combi Fluo-ATR probe was designed, fabricated and tested. Diffuse NIR-reflection and fluorescence spectroscopy as the fastest measurement techniques were used for investigation of surgically removed colorectal tissue samples in a few minutes after resection. Taking into account the necessity of rapid measurement process, we developed multispectral NIR-UV probe that enables efficient excitation and collection of NIR diffuse reflectance and fluorescence spectra of the same point. Needle probe design with diameter 0.7 mm allows to penetrate in some depth of tissue and obtain most reliable spectra avoiding artefacts related to surface drying. Multivariate data analysis proved the increased sensitivity of methods combination in one dataset (94% Sensitivity vs 85% or 85% for fluorescence or NIR reflection correspondingly).

A multimodal method for skin cancer diagnosis based on Dermatoscopy (DS) image analysis, Hyperspectral Imaging (HSI), Raman/Autofluorescense analysis (RS/AF) and Optical Coherence Tomography (OCT) is presented. A universal multi-class classifier for OCT is described. The diagnostic accuracy near 90% (depended from tissue type) has been demonstrated for in vivo and ex vivo cases. A multimodal unit for diagnosis of cutaneous malignant neoplasms has been proposed. It include following pure optical modalities: Raman Spectroscopy (NIR, 785 nm) in combination with Autofluorescence analysis (485 and 785 nm), Optical Coherence Tomography (840±45 nm) and Hyperspectral Imaging (visible range, 450-750 nm, 2.3 nm spectral resolution). The method is also supplemented with a Multispectral Digital Dermatoscopy, which is a perfect solution for preliminary screening and diagnosis of pathological areas by general physician. The own developed DS tool is based on the Basler acA1920-25uc camera (RGB, 12 bit/px, 1920*1080), its resolution on the surface of the skin is about 13 μm/px. The device body has been designed and printed on a 3D printer. Tissue lighting is carried out by various LEDs: UV 365 nm, White 4000 K (polarized and unpolarized LEDs); Blue 465-485 nm; Green 520-535 nm; Red 620-630 nm LEDs. The results of several in vivo and ex vivo series of experiments are presented. In vivo studies were performed in Samara Oncology Clinical Center. A total of 220 patients were examined with Malignant Melanoma (MM), Basal Cell Carcinoma (BCC) and other benign and malignant tumors. 232 tissue samples surgically resected from patients were examined ex vivo. All samples diagnoses were confirmed by histology reports after resection. A final accuracy of 97.3% was obtained using RS/AF analysis. Moreover, for case of Malignant/Benign in vivo case, it reached up to 100% with low-cost Ocean Optics QE65 Pro spectrograph and PLS analysis. A final separation of classes for OCT ex vivo leads to a high (95%) accuracy for two-classes cases and 75% for the four classes case. Combination of 2D and 3D OCT data give us an opportunity to receive common information about tumor (geometric and morphological characteristics) from data cubes and use more powerful algorithms for counting features (fractal, textural, geometric) on separated scans. These categories of features provide closer connection with ABCDE criteria (asymmetry, borders irregularity, color, diameter, evolution). Final accuracies 90% for in vivo HSI is very similar to in vivo DS accuracy, which reaches 93%. The optical density spectral feature has been used as a main diagnostic characteristic for HSI and topology texture and color analysis for RGB DS.

The most valuable results for the use of OCT imaging in ENT diagnostics have been shown by Stephen Boppart and his group in Illinois Urbana-Champaign University. In 2016 this group demonstrated the possibility to reconstruct some viscosity properties of the effusion by the use of OCT providing some additional measurements. Our team have provided the pilot study on the possibility of detection of the effusion using the OCT device in 2014 13. The current work is devoted to improvement of Dr Boppart’s approach to examine the effusion viscosity. To provide the preliminary investigations we used the time-domain OCT device due to the reason of its clinical approval. This device provides about 200 A-scans per second, which is quite enough to register the Brownian movement of the scatterers in the middle ear effusion. All investigations were made by the use of thin (2.4 mm diameter) flexible forward-viewing probe 14 pushed through the standard ear mirror. The main disadvantage of the probe is the requirement of the contact between the tip and the tympanic membrane, which may cause some discomfort to the patient. In the order to enhance the image brightness behind the tympanic membrane the immersion was injected in the auditory meatus. The use of immersion also provides the reference level of scatterers mobility while the effusion viscosity was examined. The conventional OCT image of the eardrum demonstrates the high level of backscattering particles behind the membrane in the case of otitis media with effusion. Following 11, we switch scanning off and recorded images. The obtained image became time-resolved and moving scatterers are presented here as quite short horizontal lines on the image while the steady area is presented by long horizontal lines set. Dr Monroy used time-correlated analysis to estimate the mobility of particles suspended in the effusion. We propose to use the Fourier analysis of the image, which seems to be more informative. We implemented the 2D-Fourier transform to the OCT data recorded while the scanning was switched off. One can easily note the differences in the width of the spectrum between areas of eardrum, water immersion and effusion noting the effective spectral width. To numerically estimate the width of the Fourier image we fit every row data with the Gaussian shape. After that the FWHM parameters of the Gaussian shapes were used to obtain the in-depth profile of the particles “mobility. One can see that the obtained by proposed method particles “mobility” is quite different in cases of the water suspension and the effusion. We have proposed the method of estimation of the middle ear effusion viscosity using the Fourier analysis of the OCT data obtained by the fixed probe beam. The method provides the possibility to distinguish areas filled by water and effusion. We believe this approach to be useful in differentiation the grade of the otitis media with effusion in clinical conditions. This research was supported by Russian Science Foundation (project No 17-15-01507).

In this paper, a surface Plasmon resonance (SPR) based photonic crystal fiber has been proposed and numerically investigated by Finite Element Method (FEM). The proposed SPR-based PCF shows higher average wavelength interrogation sensitivity than the previous structures. Different plasmonic materials have been used to show the difference in results. Liquid filled cores with metallic surface can be exited with leaky-Gaussian core guided mode. Numerical investigation of optical properties for the proposed PCF has been established by changing the designing parameters like pitch, diameters etc. The proposed PCF is simple in nature and can be easily fabricated by existing methods. Biological substances, biochemical, organic chemical analysis, bimolecules can be detected by our proposed SPR based PCF.

A new laser method for increasing uveoscleral outflow path for normalization of intraocular pressure in glaucomatous eyes is presented.

Nonuniform laser heating affects the porous system of biological tissues. Formation of new pores in the paralimbal region of the eye can accelerate the flow of the intraocular fluid through the eye sclera and, thus, facilitate normalization of the intraocular pressure. A positive effect of laser impact is achieved, as a rule, in a narrow range of laser radiation parameters, which makes it difficult to choose the intensity and time parameters of laser irradiation due to such factors as nonstationary temperature fields, thermotensions and pressure that can give rise to undesirable effects and complications.

The comparison between reflected and transmitted laser light through the eye tissue has allowed to establish the main requirements for laser settings parameters responsible for efficacy and safety of the laser irradiation. The positive effect is achieved only by using relatively small intensity of the laser radiation. At high intensity, the hydraulic permeability decreases due to denaturation and tissue hardening.

Atomic Force Microscopy (AFM) measurements with nanoindentation and optical coherence tomography (OCT) based compressional phase-sensitive optical coherence elastography (OCE). OCE measurements demonstrated laser-induced dilatation areas attributed to formation of ensembles of micro-and nano-pores in sclera providing increase in its hydraulic permeability. Much higher resolution AFM examinations directly demonstrated such individual irradiation-produced pores. At the same time, the collagen structure of the sclera is not destroyed, and tissue mechanical properties do not degrade under laser radiation. The process of pore formation is in good agreement with computer simulations of the dynamics of thermal stress fields induced by laser irradiation.

Volumetric texture analysis method was developed for tumor segmentation of 3D optical coherence tomography (OCT) images. Images were divided into small volumes of interest (VOI) around each voxel. OCT speckle intensities from those VOIs were plotted as histograms and fitted with gamma distribution function to obtain curve shape (alpha) and scale (beta) parameters. Alpha/beta ratio 3D parametric images clearly delineated tumor and normal tissues otherwise not separable in structural OCT images. Method was validated with confocal fluorescence microscopy, tumor cells fluorescence and histological staining. Volumetric variation of OCT speckle intensities from same datasets was used to obtain microvascular information. Method was tested on three different tumor types (melanoma, cervical carcinoma and pancreas adenocarcinoma) in two mouse models: a) Nude mice with B16F10 pigmented murine melanoma tumors grown under the skin; b) NRG mice with human ME-180 and Bx-PC3 tumors grown in dorsal skin window chambers. Combined alpha/beta and microvascular images for all three tumor types demonstrate robustness of the method for detection of tissue variability and separation of tumor and normal tissues.

Widefield optical coherence tomography (OCT) can be used to image the posterior segment of the human eye. OCT images are typically distorted due to the inconsistent scanning and display geometry and the refractive properties of the human eye. The goal of this study was to create and validate an estimation model that maps the OCT images to the true geometry of the human eye. In addition, we wanted to assess how refractive parameters of the human eye and instrument-to-eye alignment errors affect the estimation model. We have developed a model that estimates the true curvature of the posterior human eye based on widefield OCT images. We have experimentally validated the estimation model using two different phantoms, a single refractive surface solid glass test eye with a spherical retina, and a waterfilled test eye with anatomically correct cornea, lens, iris, and retina. In order to further evaluate the suitability of the model to estimate the shape of the human eye, we have performed a tolerance analysis of the critical alignment and refractive parameters in the model, including axial length, corneal power, refractive indices, and lateral and axial alignment of the eye relative to the OCT system. We have found the estimation model to be highly sensitive to variations in axial length and less sensitive to variations in working distance, corneal power, eye alignment, or refractive indices. In conclusion, we have demonstrated an estimation model that estimates the shape of the human eye based on widefield OCT imaging. We further conclude that we can appropriately estimate the shape of the human eye based on widefield OCT images by using nominal values for the refractive properties and actual measurements of the axial length of the eye.

A swept-source optical coherence tomography (OCT) system is demonstrated in the mid-infrared region. A Michelson interferometric setup is illuminated by an external cavity quantum cascade laser (QCL), with a scanning frequency of 1 Hz. A-scans were collected using three different samples: a mirror, CaF2 coated with germanium on both of its surfaces, and CaF2 coated with germanium on the back side of the sample. These depth-profiles were used to mimic a tissue sample with multiple reflective boundaries. Fourier transformation of these interference fringes clearly showed the expected depths of reflection, allowing for the signal to noise ratio of the system to be determined.

Urothelial carcinoma (UC) is the most common type of bladder cancer. The gold standard for detecting UC is white-light cystoscopy, which is followed by tissue biopsy and pathological examination. However, such process is invasive, timeconsuming and prone to sampling errors. In this framework, optical spectroscopy techniques provide fast, label-free and non-invasive alternatives to standard histopathology. Thus, the aim of this study is to discriminate normal bladder tissues from urothelial tumours, and to identify the different stages of the disease, by means of combined auto-fluorescence, diffuse reflectance and Raman spectroscopy. In fact, these techniques were implemented in a compact and transportable setup based on two optical fibre probes: one coupled to fluorescence and reflectance excitation sources, while the other one to the 785 nm laser. Raman, fluorescence and reflected light signals were collected though the same probe used for excitation and sent to a spectrograph. We used this experimental setup for studying fresh biopsies of urothelial tumour and healthy bladder collected from 32 patients undergoing Transurethral Resection of Bladder Tumours (TURBT). Scoring methods based on ratiometric approach and Principal Component Analysis (PCA) allowed not only to discriminate healthy biopsies from tumour ones, but also to recognize three tumour stages.

The diffuse correlation spectroscopy (DCS) and diffuse near infrared spectroscopy (DNIRS) are the contemporary non-invasive optical methods which have turned out now to be ones of the most required optical tools for assessing tissue health, in regards to mammography, brain, and deep tissue injury. Earlier we reported on an observation, within the DCS technics, of development of pressure injuries measuring dermal and subcutaneous red blood cell motion; the data obtained has produced remarkably a characteristic decay time of the light intensity temporal correlation function being five times larger for patients of the group with developing open pressure injuries as compared with the group exhibiting healthier stage. The quantitative determination of the characteristic time required a definite picture of scatterer motion. For quantitative study the crucial problem to solve is a proper account for the scattering anisotropy. We perform comparative simulations of the diffuse photon density wave (DPDW) signals and the temporal intensity correlation functions either with the Henyey-Greenstein (HG) or Rayleigh-Gans (RG) phase functions, which we consider is more appropriate as the hard sphere suspension model for imitating a tissue. We find that for a half space geometry the results obtained for these two scattering patterns turn to be quite close; however for finite size tissue geometries results of simulations of the source-detector plot for backscattered intensity differ noticeably at small distances; simulating the temporal correlation function with these two phase functions we find the blood flow to be different for different scattering patterns in case of spatial restrictions. The DPDW methodology is widely used in a number of biomedical applications. Here we present results of Monte Carlo simulations that employ an effective numerical procedure, based upon a description of radiative transfer in terms of the Bethe-Salpeter equation, and compare them with measurements from Intralipid aqueous solutions. We find the Monte Carlo simulations and measurements to be in a very good agreement for a wide range of source-detector separations.

This paper discusses the resolution required to provide a ‘new affordable generation’ of ophthalmic instrumentation for imaging the retina. The paper describes the evolution of the direct ophthalmoscope as a device that can capture digital images of sufficient detail and contrast such that they both medically useful and can form a wide-field composite image of the human retina comparable to that of a fundus camera.

During the early stages of diagnosis, medical practitioners often rely on an ophthalmoscope for making a first inspection of the retina. It is an instrument which has taken considerable time to change from human observation to a digitally processed image. There is a good reason for this, the instrument relies on the near to diffraction limited imaging ability of the human eye and the ability of the user to scan the instrument over the retina to create a high resolution contiguous mental image. Whereas the more sophisticated instruments such as the fundus camera relies on conventional imaging technology to make a digital imaging record of the retina. The digital ophthalmoscope described creates an image with 5μm resolution over the whole retina.

This paper discusses the comparison of the of resolution quality which can be achieved using digital storage of retinal imaging. Two devices will be considered the direct ophthalmoscope, which is essentially a hand-held portable device for direct inspection and the fundus camera. The analysis argues that currently the resolution of digital camera technology used in the fundus camera, particularly those used for mobile scanning, limits its optical diagnostic power. Whereas, a digital ophthalmoscope using well tried imaging stitching software and digital processing provides an alternative higher imaging resolution, hand-held portable alterative.

Enormous potential of nanoparticles in medicine is a rapidly growing research field. Hereby, we focused on the applications of biocompatible oxide nanoparticles in the field of cancer diagnosis and therapy. This work was focused on the development of fluorescent Tb-doped ZrO₂ nanoparticles (NPs) for application in lung cancer diagnostics. Obtained, hydrothermally created NPs were below 100 nm with very low influence of Tb concentration on size. Mice received suspension of nanoparticles (10 mg/ml, 0.3 ml/mouse) via gastric gavage. All protocols were according to the EU guidelines and approved by LEC agreements No 2/2012 and 13/2015. At 3h and 24h mice were sacrificed and all tissues collected for analyses under confocal microscope and scanning cytometry. Following oral administration, ZrO₂:Tb nanoparticles were passively targeted to all tumour loci via the enhanced permeation and retention (EPR) effect. Due to the very tight endothelial barrier in the lungs NPs in this organ were targeted specifically to the areas of metastases rendering them a highly specific diagnostic tool for cancer diseases with high potential applications as a carrier of therapeutic factors.

The mechanical properties of biological tissues are increasingly recognized as crucial parts of signaling cascades involved in developmental and pathological processes. Most existing mechanical measurement techniques require either highly invasive sample preparations and destruction of the tissue for access, such as atomic force microscope, or provide insufficient spatial resolution, such as sonoelastography and magnetic resonance elastography. The optical elastography is an emerging field in biomedicine, which allows to capture an image of the elasticity module with subcellular resolution. We present as a promising method a quantitative micro-elastography based on Brillouin scattering, which is the inelastic scattering of photons by acoustic phonons with gigahertz frequency. Using a virtually imaged phased array (VIPA) based spectrometer and a confocal microscope a label-free, three-dimensional, non-intrusive micro-elastography with the absence of extrinsic mechanical loading is provided. In this paper, we present a systematic application of Brillouin micro-elastography to quantify physical properties of native larval zebrafish tissues in vivo. We detected a transiently decreasing Brillouin frequency shift after spinal cord injury. The presented work constitutes the first step towards an in vivo assessment of spinal cord tissue mechanics during regeneration, provides a basis to identify key determinants of mechanical tissue properties and allows to test their importance in combination with biochemical and genetic factors.

In this contribution, combined variational mode decomposition (VMD) aided non-linear feature descriptors & artificial neural network (ANN) for identification of different healthy and precancerous cervical tissues. Owing to the inherent problems of background laser system noise interferences in elastic scattering spectroscopic data, VMD method being noise robust is of paramount interest. VMD is used to decompose the normalized spectral data into 2 modes for analysis and attributes extraction. For each of these VMD separated modes, non-linear entropy and multifractal features, namely Shannon entropy (SE), Renyi entropy (RE), Tsallis entropy (TE) and Singularity spectrum width (SSW) are extracted to form the feature set. The extracted features are subjected to analysis of variance (ANOVA) test for subsequent feature ranking & selection of the statistically most significant features. The designated features are trained with ANN to classify the backscattered tissue spectra into healthy and cancerous ones.

Assessment of skin tissue perfusion is vital for understanding the normal and the pathologic physiology of human body. Diffuse optical methods provide numerous pathways for assessing various static and dynamic perfusion markers such as variations in bulk tissue optical properties, depth dimensions of microvascular bed, rate and volume of blood flow and so on. There have been numerous studies on these aspects ending up with qualitative assessments on various parameters, where separate approaches are explored for individual parametric evaluation. With the introduction of precise optical tissue phantom models, integration of different static and dynamic perfusion markers are possible to facilitate quantitative assessment of such a perfusion matrix. In this work, we present the fabrication of a perfused tissue physical model that mimic skin tissue and subsequent estimation of perfusion matrix including optical properties, flow, and depth of the microvascular bed. Different layers of skin are spin coated onto micron-sized embedded channels, and the model was subjected to optical measurements, inducing different flow levels using a syringe pump. The parameters have been estimated using spatially resolved diffuse correlation optical spectral measurements, using a handheld fiber optic probe with a precise source to target distance sensing mechanism and associated signal processing algorithms. This work is aimed to provide a methodology for quantitative assessment of various perfusion parameters using a versatile physical model that provides flexibility in varying involved parameters accurately. The work performed here, after standardization is expected to have potential in developing non-invasive quantitative optical skin biopsy tools to augment the current histopathological studies.

In the experiment, the reaction of the rat oral mucosa to fractional mechanical and laser treatment performed by radiation of a diode laser with a wavelength of 980 nm in various combinations of the average power of laser radiation, the pulse duration and energy of the laser radiation, along with the fill factor was investigated. Optical and histological examination of the rat oral mucosa during its regeneration after fractional treatment was performed. It was found that on the 5^th day after a single-shot fractional laser treatment, the number of mitotically dividing cells exceeds the number of those cells after a single-shot fractional mechanical treatment and in the control area. It has been found that on the 28^th day after a three-fold fractional treatment, the surface of the mucosa is visually bleached, and after the laser treatment, the connective tissue of the mucosa lamina propria and the submucosal layer contains fibrous structures and cellular elements, mainly myo- and fibroblasts. It has been found that during the regeneration on the 28^th day after a three-fold fractional laser treatment, the thickness of the epithelium and the thickness of the submucosal layer with lamina propria of mucosa increase in comparison with the control.

We have proposed a concept of monitoring ice ball formation in biological tissues during cryodestruction process via spatially-resolved detection of elastic light backscattering. For this purpose, we developed an experimental setup for study cryodestruction by using applicators based on sapphire shaped crystals with internal channels for optical irradiation of biotissues and detection of backscattered light. Due to the unique physical properties of sapphire, i.e. high thermal, mechanical, and chemical strength, high thermal conductivity and optical transparency, the sapphire cryoapplicators yield combination of the tissue cryodestruction with the optical control of tissue freezing. We have shown experimentally that using the proposed concept of applicator with several channels, it is possible to monitor changes of the ice ball during the cryodestruction process.

In this paper, we investigated ¹⁸F-THK5351 PET/CT image of normal Thai population for the optimized time of radioactive tracer PET/CT. Twenty-five volunteers without neurological or psychiatric illnesses and all of them have no abnormalities detected on neurologic examination. All subject were underdiagnosed on ¹⁸F-THK5351 PET/CT and 3.0 Tesla MR imaging. THK5351 PET/CT were operated on the co-registered MRI comprised for drawing an ROI. The DICOM file was converted to .img file, .hdr file and then merge with each other for obtaining an fmridata.mat. Position of ROI and fmridata.mat were used to plot graph showing the relationship between quantitative uptake with the frame of image. The optimized time of ¹⁸F-THK5351 PET/CT in normal Thai population is 50 to 70 minutes.

Worldwide, outdoor air pollution is responsible for 4.2 million premature deaths per year. Both chronic and acute exposure to particulate matter air pollution is a risk factor for heart and lung diseases. One of the atmospheric pollutant particles is represented by soot or carbonaceous particles (CPs), which are produced during the incomplete combustion of fuels. To evaluate human CP exposure, a direct and label-free approach for detecting such particles in body fluids and tissues was still lacking. We present a novel technique to finally close the diagnostic gap. We report for the first time white-light generation by CPs under femtosecond pulsed near-infrared light illumination in aqueous environments and demonstrate the potential of this approach in biomedical and diagnostic context. In fact, it was shown that urinary carbon loading can serve as an exposure matrix to carbon-based air pollution, reflecting the passage of soot particles from circulation into urine. The novel method is straightforward, fast and flexible without the need of sample pretreatment. Moreover, the technique offers several other advantages such as inherent 3D sectioning and high imaging depths making it possible to screen at the cellular and tissue level. In conclusion, this novel diagnostic technique allows to quantify exposure at the personal level including different scenarios like occupational exposure, smog, forest fires, etc.. Additionally, this approach paves the way to unravel the complexity of soot-related health effects.

Video-based heart rate estimation based on the PPG technique is a remote optical technique allowing to determine the heart rate and breath rate through the intensity or motion variations. This paper proposes a new monitoring method for simultaneous estimation of heart and breathing rates using an active 3D system with structured light. Active 3D imaging systems capture information about 3D object shapes using artificial illumination. An active 3D imaging system can be realized using different techniques: time-of-flight, triangulation, motion flow, interferometry. In this work, we investigate triangulation-based systems. Heart and respiration rates are signals analyzed in proposed systems as crucial vital signs from a medical point of view. Furthermore, the type of respiration is also analyzed with separation between abdominal and torso type. The quality of breathing signals received from various optical systems is also compared. This system is based on different approaches to 3D imaging and includes random light pattern, fridge light pattern and active stereo methods. In the light pattern systems, the movements of the chest and abdominal are associated with breathing and are observed by the video camera as the light pattern deformation. The system based on active stereo method provides the depth map by which the breath signal is extracted. Further analysis of received signals is based on Principal Component Analysis (PCA). The proposed remote optical technique extracts the blood pulse from an image captured by a vision system. Patient’s face is detected with use of dedicated convolutional neural network for robust face alignment. Based on found features points, a few regions of interest are selected, and then their results are compared. Mean pixel values for three color channels (red, green, blue) are calculated. Active 3D imaging technique enables stabilization and quality improvement of the captured image. Not correlated groups of pixels are eliminated. Then cluster analysis is used on the grounds of multi-features. Measurement data are detrended by empirical mode decomposition EEMD. Subsequently, ICA was applied to recover source signals from the observations. Maximum frequency pick in the FFT spectrum determines HR. The performance of the proposed system was tested through a series of experiments. Vital signs of 10 persons with reference signals have been detected. Both, breath and heart rates estimates were found to be accurate. The proposed system can be used as a component of more complex medical systems where information about the condition of the examined patient is crucial or important.

Optical fibre based endoscopes are increasingly used for imaging and sensing within the human body without navigational guidance of the miniaturised fibre probe. Meanwhile, other medical device placement is a standard procedure in clinic. We demonstrate successful imaging of optical device location with centimetre resolution in clinically relevant models, in a realistically lit environment, achieved through the detection of early arriving photons with a time resolved single photon detector array. This prototype has been developed within the UK-EPSRC Proteus project, moving advanced research technologies towards clinical implementation. Short (~100ps) laser pulses are transmitted from the tip of the endoscope at 785nm in the “optical window” where attenuation is less severe in clinical scenarios. Most of the photons that pass through tissue undergo much scattering from the disordered tissue structures providing only low accuracy determination of the location of the light source. However, some photons probabilistically undergo less scattering, travelling through the medium in an almost straight line without a much extended path. Such photons exit the body sooner than the highly scattered light. A camera based upon a 32 × 32 array of Single Photon Avalanche Diodes (SPADs) made with CMOS technology is used to image the small number photons exiting the tissue. The time resolution capabilities of such a single photon detector (50ps time bin resolution, 200ps jitter) allow observation of the photon arrival times simultaneously for all 1024 pixels of the imaging array. Photon arrival statistics distinguish the early arriving photons from the highly scattered light, revealing the endoscope location. Scattered photon arrivals peak at delays of multiple nanoseconds due to the thick tissue samples. The progression of light through complex scattering structures can be observed. Normal fluorescent room lighting has distinct emission peaks. Appropriate choice of operating wavelength between these spectral features, combined with aggressive filtering, allows operation in normal fluorescent lighting. This compact packaged system is demonstrated in a normally lit room to determine optical endomicroscope location in a whole ventilated ovine lung as well as tissue models including bone structure. At the limit of capabilities of this prototype, demonstration through an entire human torso is shown to be possible. System improvements and the potential of the next generation prototype in development will be discussed. This offers the potential for real time (sub second) imaging of device location with a portable system for application in standard medical procedures, such as catheter insertion. The avoidance of the need to confirm device placement with X-ray imaging has potential to decrease disruption to procedures throughout clinical practice.

Immune diseases are associated with the activity of cellular and humoral immunity. The humoral (molecular) immunity plays the role in mechanisms of immunological tolerance and pathogenesis of many diseases, including cancerous. The opportunities as well as the challenges facing the humoral immunity analysis are formidable. The study of molecular composition and functionality of humoral immunity is capable to reveal effective strategies for early diagnosis and treatment. The optical techniques were selected for this research due to the noninvasiveness and simplicity of in vivo imaging, allowing real-time observations of the immune activation process. The laser correlation spectroscopic technique allows one to determine sizes of nanoparticles, in particular molecules and molecular agglomerates. In this work, the original scheme of the device based on laser correlation spectroscopic technique is suggested. In the presented work, the molecular compounds were investigated in blood serum. We measured the size distribution of protein molecules in blood of several donors. We observed both single proteins and the protein agglomerates demonstrating normal course of metabolic process in an organism. Then we initiated the immune reactions by the addition of the vaccines to the blood serum. After that, the noticeable aggregation of proteins was observed. To separate specific and nonspecific immune reactions a blood serum with inactivated complement system was also studied. It showed a significantly different outcome. So the laser correlation spectroscopic technique can be successfully used for analysis of the immune activation processes and further diagnostics.

Time-resolved vibrational spectroscopy is an important tool for understanding biological processes and chemical reaction pathways [1]. Today, all available methods to our knowledge require many repetitions of an experiment to acquire a microsecond time-res. mid-IR spectrum. We present the IRspectrometer, a quantum cascade laser dual frequency comb spectrometer [2-3]. It allows for parallel acquisition of hundreds of mid-infrared wavelengths with microsecond time resolution. The formation of the light-activated L, M and N-states in bacteriorhodopsin – which only have µs to ms lifetimes – has been recorded that show the infrared response of bacteriorhodopsin to 10 ns visible light pulses with microsecond time-resolution. The different wavelengths were all measured in parallel thanks to the dual-comb approach. The spectra as well as the kinetics show good agreement with those from step-scan FT-IR measurements. As a benchmark, the spectral signature of several intermediate states of the bacteriorhodopsin photocycle has been recorded in a single shot measurement. This approach greatly reduces the complexity of time-resolved bio-spectroscopy measurements in the mid-infrared which currently require many repetitions.

Protein modification is the process by which the order of amino acid sequencing is rearranged or the functional groups on the amino acids are altered by a chemical process. Protein modification is responsible for processes such as metabolism, metastatic behavior, mental cognition and muscular movement [1]. In order to understand protein modification through amino acid alteration, it is first necessary to understand the real time behavior of the amino acid groups under stress as well as develop a method to analyze multiple amino acids within a single sample. Our goal was to analyze the resilience of the amino acids and to understand the impact their presence would have on the specificity of the Raman bands that indicate spectral fingerprint or vibrational modes. A blend of three branched chain amino acids, L-leucine, L-valine and isoleucine, were investigated for stress analysis and strain effects due to local heating of the sample through confocal Raman spectroscopy and compared to individual amino acid Raman spectroscopy. Data was collected using a full factorial design of experiment (DOE) with the factors including laser power, acquisition time, and spectral range, where each was repeated three times. With a laser spot size of approximately one-micron in diameter, the laser power and acquisition times were varied over the spectral range of 50-3600 cm^-1. The crystal size of the blended samples were between 5-10 μm in width and 10-15 μm in length. The crystal size of the individual L-leucine and L-valine samples were between 20-40 μm in diameter. The individual isoleucine samples were between 15-20 μm in width and 60-80 μm in length. Assignment of each band in the blend derived from previously reported bands for the respective amino acids, where L-leucine influenced the majority of the Raman bands observed. The spectra collected from the blended sample were compared to the individual spectra collected, and when the laser irradiance was increased, local heating effects gave rise to bands stemming from the Lvaline and isoleucine indicating the relative resilience of the individual amino acids. Our samples yield high spectral response in the 2800-3100 cm^-1 range owing to the C-H bonds of each component. This is expected as our samples were comprised of three-branched chain amino acids with functional groups comprised solely of C-H bonds. When analyzing a multicomponent amino acid sample where characteristics of the minor constituents are desired, Raman spectroscopy can play a significant role in revealing the underlying structure. Principal component analysis (PCA) was performed using each of the individual spectra and the blended spectra. PCA showed the first coefficient for Lleucine was responsible for approximately 15% of the blended spectra.

Optical spectroscopy has proven to be a very powerful method in bio-photonic technologies. The multimodal use of different spectroscopic techniques makes this approach especially promising for medical diagnosis. For preventive diagnostics, there is great interest to use as much information as possible regarding the tissue before, during and after treatment. Spectroscopy can non-invasively assess morphologic and biochemical changes, thereby ensuring pre-treatment identification of tissue type and discrimination between healthy and diseased tissue. It is ideally suited for molecule-specific recognition of biomarkers and tracking treatment progress. The study is focused on the research, development, design and tests of a novel system based on Raman, Near-Infrared (NIR) and Fluorescence spectroscopy merged with temperature measurement for diagnosis of different diseases, i.e. cancer. Combination of all these methods together with multivariate signal and data processing makes the device universal and leads to an increase of diagnostic accuracy, sensitivity and specificity. The compact, cost-effective, portable and modular design is the main advantage of the combined spectroscopic system. Overall dimensions of the system measure only 30×23×20 cubic centimeters. It consists of 3 spectrometers (Raman, Fluorescence and NIR) and 3 light sources (785 nm laser for Raman, 532 nm laser for Fluorescence and halogen lamp for NIR) for spectroscopic methods plus a pyrometric temperature sensor. All modules are equipped with specially designed fiber-optic probes (SiO2 fibers for the spectroscopy and PIR fibers for the temperature sensor) for increased flexibility. The software is realized by a personal computer (PC), requiring only one graphical user interface for hardware operation and data processing. This allows measurement and results within seconds. The system may function independently of a PC as a stand-alone tool, using an installed microcontroller, which permits the processing functions. System modules were tested separately and together on different types of chicken, bovine and porcine tissue. Furthermore, several ex vivo biopsies of healthy or cancerous human tissue and bio-liquids (serum, plasma, urine) were analyzed. Temperature sensing was tested in real-time, monitoring the local temperature changes seen in laser surgery.

An experimental setup for super-resolution microscopy by structured illumination is presented, preliminary experiments of nano-beads and living cells with a resolution around 100 nm are described, and further requirements for live cell microscopy are discussed.

In recent years nanotechnology gathered much attention due to promising applications in biomedicine. Using nanoparticles as drug carriers could allow for more effective and efficient therapy in treating cancer or neurological diseases. This is due to their unique properties such as enhancement of drug bioavailability or the ability to protect the drug from degradation. In this study we performed in vivo (BALB-c mice) and in vitro (Caco-2 cell line) experiments with Y₂O₃:Tb_lectin conjugates as well as pure lectin to characterize the dynamics of nanoparticles mediated drug uptake from gastro-intestinal tract. Mice were given 0.3ml of Y2O3:Tb_lectin conjugates or pure lectin suspension and were sacrificed after 3h, 24h and 1 week (Y₂O₃:Tb_lectin conjugates) or 3h and 24h (pure lectin). Cell cultures were incubated for 24h with increasing concentration (0.001mg/ml; 0.01mg/ml; 0.1mg/ml; 1mg/ml) of Y2O3:Tb_lectin or pure lectin. After analysing gathered data we concluded that our nanoparticles successfully conjugated with lectin and allowed for its transport through physiological barriers. NPs_Lectin conjugates undergo absorption, distribution and redistribution similarly as free nanoparticles do, although it decreased the efficiency of absorption compared to free nanoparticles. Lastly after reaching the tissue conjugates dissolved leading to lectin deposition in the tissue.

In the present work the possible benefits of implementation of polarization sensitivity with synchronous fluorescence spectroscopy for differentiation of ex vivo colorectal tissue samples was evaluated. Synchronous fluorescence spectroscopy (SFS) is superior method for fluorescence analysis in comparison with standard fluorescence spectroscopy. Parallel and perpendicular linear fluorescence polarization, in respect to the polarization of the excitation light, was additionally applied with SFS. Same parameters based on the area of the most prominent fluorescence maxima for SFS without polarization, with parallel and perpendicular polarization, were compared. The introduced polarization sensitivity improves the statistically significant difference of some of the evaluated parameter for cancerous and healthy tissues and their potential application as diagnostic criteria.

Magnetic Resonance Imaging (MRI) is considered a useful non-invasive method for cancer detection. However, MRI still has some limitations: low specificity for early-stage cancers as well as toxicity of Gadolinium ions, which were reported to accumulate in the nerve tissue and kidneys. Early cancer development and metastases monitoring are still difficult, because of the issues with permeability of contrasting agents through the blood-organ barriers. Nowadays, studies are being conducted to find the new contrasts with high magnetic moment, yet without gadolinium-induced toxicity. We propose an innovative, multimodal, high-k oxide-based contrasting nanoparticles (NPs), combining fluorescent properties of lanthanides with contrast in T1 and T2 spin relaxations. This material can facilitate both in-situ screening and visualization of tumour for fluorescence assisted biopsy or surgery. NPs used in our study were developed in the Institute of Physics, PAS. The NPs core was based on HfO2, doped with Eu ions, while Gd was used for positive control. Fluorescence was induced at 619nm, while emission was detectable at 630-650nm. The T1 and T2 relaxation times have been assessed using phantoms. Statistically significant changes were observed in T2 relaxation time. We used old rats, patients of the oncology clinic as an animal model. Prior to oral application of NPs (1mg/ml, 1ml/rat, LEC No 13/2015) the initial MRI screening of rats was performed. Weighted images T2 (3D FSE), SWI and SS-FSE were performed twice, 24 and 48 hours after IG. After imaging, tumours were surgically removed, for cytometric and pathomorphology evaluation.

Introduction. Fluorescence spectrometry allows studying skin autofluorescence (AF), which shows content of advanced glycation end products. The aim of study was to determine possibility of using the experimental sample of fiber optic spectrometer FOS-1 for diabetes mellitus diagnostics and control, including noninvasive diagnostics of complications.

Methods. We involved 36 healthy participants, 13 type 1 diabetic patients, 10 type 2 diabetic patients. The 1st and the 2nd groups were comparable by the age, gender, skin reflection coefficient, characterizing skin phototype and degree of tanning. Skin AF was measured at a wavelength of 460 nm with excitation of 365 nm. To reduce effect of skin pigmentation, ratio of fluorescent signal to signal of reflection in excitation region was used as measured parameter.

Results. Significant correlation between AF intensity and age was found in type 1 diabetic and control groups (R=0.6, р<0.05 and R=0.43, p<0.05, respectively). No significant difference in AF level was found between these groups: median AF was 0.87 (0.86; 0.89) arb. units and 0.85 (0.77; 0.88) arb. units respectively. In type 1 diabetic group AF also positively correlated, although not statistically significantly, with diabetes duration, glycosylated hemoglobin level, average daily glucose level (R=0.52, p=0.06; R=0.45, p=0.09 and R=0.56, p=0.07 respectively). The median AF was 14.7% higher (p=0.34) in patients with several diabetes complications than in diabetics with 1 complication and 13.9% (р=0.19) higher than in patients without complications.

Conclusion. Obtained data show possibility of using the described method with spectrometer FOS-1 for diabetes control and for diagnostics of microvascular complications.

Nanotechnology as a new field of science is broadly exploited in a plethora of commercial uses. Biocompatible nanomaterials are also attractive for medical applications. However, an exact processes related with their biodistribution within the body needs to be examined. This study deals with future perspectives for biodegradable nanoparticle on the example of fluorescent ZnO NPs (zinc oxide nanoparticles), doped with europium (Eu) ions. The aim of the study was to evaluate distribution processes of biodegradable ZnO:Eu NPs within the living organism. ZnO:Eu NPs were administered intra-gastric (IG) (10 mg/ml, 0.3 ml/mouse) to adult Balb-c mice (n=35) and following 3h, 24h, 7d, 14d and 1m mice were sacrificed and internal organs were collected, as was described before [1]. For determination the excretion patterns of these nanostructure, ZnO NPs were orally administered to mice (n=24) with further measurement of zinc content in the feces of tested animals. All procedures were conducted according to local and EU regulations and approved by the LEC 44/2012. No pathological/behavioral changes were observed in mice. Biodegradable ZnO:Eu NPs revealed ability to overcome majority of physiological barriers in the organism, which renders them invaluable tool for biomedical applications. After 3h the presence of fluorescent NPs was already observed in key tissues and the peak of NPs distribution was observed at 24 h after IG in majority of tissues, including brain. Moreover, obtained results revealed fast and efficient clearance of ZnO NPs from the living organism, even following multiple administration of nanostructures (up to 4d after IG).

This talk will describe ratiometric imaging that can enhance visualisation of imaging probes against tissue autofluorescence. The Proteus project (www.proteus.ac.uk) aims to improve the detection and diagnosis of pulmonary infection and inflammation by employing targeted fluorescent molecules (Smartprobes) for labelling specific pathologies in tissue. However, imaging Smartprobes using a widefield fibred imaging system within the human lung can be challenging, in part, because both lung tissue and imaging probes have broad and overlapping emission spectra.. Weak signals from pathogens labelled with probes are easily missed due to the strong autofluorescent signatures of elastin and collagen that are abundantly present in the human lung. In addition to resolving probes from intrinsic fluorescence, multiple probes may have overlapping emission spectra themselves. This is particularly true for many well-established fluorophores that reside in the green region of the spectrum. If imaging with fluorophores that have distinct emission spectra is not possible or desirable, then spectral sorting of the signals can be carried out. To successfully resolve probes from healthy tissue or to resolve similar probes from each other, acquiring full spectral information is not necessarily a requirement. We describe a simple widefield fibred imaging system consisting of a single colour LED illumination source (480nm) that enables ratiometric methods to enhance contrast between different fluorescent sources. Fluorescence from 480nm excitation of tissue as well as Smartprobes present on the tissue is split into two optical paths, above and below a cut-off wavelength, by a dichroic mirror. A triggered system of a monochrome CMOS camera and optical chopper allows collection of dual images of the same field of view from different parts of the spectrum. Contrast enhancement is carried out by post processing of the images, enabling us to interpret better the images produced both in autofluorescence and molecular imaging contexts. Our widefield fibred imaging system is enabled by a novel optical fibre bundle developed by the University of Bath. The imaging fibres consist of 8100 cores with a 450µm corner to corner field of view and allows for multiplexed visualisation of pathologies within the lung. Biological targets, such as bacteria, that are of interest to clinicians, occupy one or two cores within the imaging fibre. We use 6µm Inspeck microspheres to demonstrate that the technique is shown to be able to distinguish targets analogous to bacteria. Also presented and demonstrated, is imaging and enhanced contrast of a biological model of labelled cells.

Cancer-associated fibroblasts (CAFs) are one of the key determinants in the malignant progression of cancer. The subject of this research was metabolic reorganization of CAFs and their participation in collagen cross-linking process. The metabolic differences between normal fibroblasts and CAFs were elucidated using two-photon fluorescence lifetime imaging microscopy (FLIM). Collagen structure in 3D model was assessed using second harmonic generation (SHG) microscopy. We show increased metabolic activity of fibroblasts derived from patient’s colon tumor with a shift to more oxidative metabolism compare to dermal fibroblasts. The results of the study of collagen suggest that CAFs may contribute to the tumor progression through the facilitation of collagen alignment. In general, our findings support the idea of the strong association between cancer cells and fibroblasts and extensive involvement of CAFs in modulation of tumor microenvironment.

Cardiovascular diseases are the main cause of death in the world and its occurrence is closely related to arterial stiffness. Arterial stiffness is commonly evaluated by analysing the arterial pulse waveform and velocity, with electromechanical pressure transducers, in superficial arteries such as carotid, radial and femoral. In order to ease the acquisition procedure and increase the patients comfort during the measurements, new optical fibre techniques have been explored to be used in the reliable detection of arterial pulse waves, due to their small size, high sensitivity, electrical isolation and immunity to electromagnetic interference. More specifically, fibre Bragg gratings (FBGs) are refractive index modulated structures engraved in the core of an optical fibre, which have a well-defined resonance wavelength that varies with the strain conditions of the medium, known as Bragg wavelength. In this work, FBGs were embedded in a commercial resin, producing films that were used to assess the arterial pulse in superficial locations such as carotid, radial and foot dorsum. The technique proved to be a promising, comfortable and trustworthy way to assess the arterial pulses, with all the optical fibre use advantages, in a non-intrusive biomedical sensing procedure. Examples of possible applications of the developed structures are smart skin structures to monitor arterial cardiovascular parameters, in a stable and reliable way, throughout daily activities or even during exams with high electromagnetic fields, such as magnetic resonance imaging.

The main purpose of this work is to evaluate the possibility to distinguish in vivo benign papilloma, severe dysplasia and squamous cell carcinoma by establishing quantitative image characteristics of multiphoton tomography (MPT) and multimodal optical coherence tomography images (MM OCT). Specific features of papillomatous outgrowths at different stages were revealed using 7,12-dimethylbenz[a]anthracen (DMBA)-induced hamster oral carcinoma. Analysis of MPT images included assessment of nuclear-cytoplasmic (NC) ratio, nuclear density and heterogeneity parameter F. Crosspolarization OCT images were quantified via the integral depolarization factor (IDF). Analysis of OCT microvascular maps enabled differential analysis based on the number of smallest-diameter blood vessels present in a particular pathology. Both MPT and MM OCT metrics showed some difference between benign papilloma, dysplastic papilloma, and squamous cell carcinoma tissue states. The results suggested that combined use of MPT and MM OCT have great potential for in vivo differentiation between benign and malignant papillomas.

We propose multimodal sensor and algorithm for automatic recognition of a food intake based on glycemic response. Embedding this sensor in a wearable device makes it possible to count number of meals at a given time and to generate personalized statistical pattern of eating habits. This pattern may have significant impact on both personal health care and big-data-driven social engineering. We use near-infrared diffuse reflectance spectroscopy, bioimpedance measurements, and binary classification for non-invasive continuous glucose trend measurements and Fourier transform based time frequency analysis of glycose trends for characterization of eating patterns and prediction of digestive system abnormalities. We tested the sensor in a series of experiments with the certain type of food and achieved 45% average accuracy of a food intake recognition with the random noise level being at 25%.

Optical Tweezers (OTs) have been widely applied in Biology, due to their outstanding focusing abilities, which make them able to exert forces on micro-sized particles. The magnitude of such forces (pN) is strong enough to trap their targets. However, the most conventional OT setups are based on complex configurations, being associated with focusing difficulties with biologic samples. Optical Fiber Tweezers (OFTs), which consist in optical fibers with a lens in one of its extremities are valuable alternatives to Conventional Optical Tweezers (COTs). OFTs are flexible, simpler, low-cost and easy to handle. However, its trapping performance when manipulating biological and complex structures remains poorly characterized. In this study, we experimentally characterized the optical trapping of a biological cell found within a culture of rodent glial neuronal cells, using a polymeric lens fabricated through a photo-polymerization method on the top of a fiber. Its trapping performance was compared with two synthetic microspheres (PMMA, polystyrene) and two simple cells (a yeast and a Drosophila Melanogaster cell). Moreover, the experimental results were also compared with theoretical calculations made using a numerical model based on the Finite Differences Time Domain. It was found that, although the mammalian neuronal cell had larger dimensions, the magnitude of forces exerted on it was the lowest among all particles. Our results allowed us to quantify, for the first time, the complexity degree of manipulating such "demanding" cells in comparison with known targets. Thus, they can provide valuable insights about the influence of particle parameters such as size, refractive index, homogeneity degree and nature (biologic, synthetic). Furthermore, the theoretical results matched the experimental ones which validates the proposed model.

Upconversion nanoparticles (UCNPs) are of interest as novel luminescent probes for numerous applications in nanobiophotonics. The small size of UCNPs enables the particles to overcome biological barriers, thereby ensuring a deep penetration into the tissues and accumulation in a number of organs. In addition, particles are known to possess high surface chemical reactivity as well as a large surface-tovolume ratio, which affect their biocompatibility. In this paper, we present data of the dark toxicity of uncoated UCNPs in the course of their interaction with tissues and organs. It was found inflammatory reaction that developing in the organism when UCNPs are introduced under the skin. The signs of damage and necrosis in all layers of the skin were not observed. The dense connective tissue capsule was formed around the particles through a week after their administration. The particles do not diffuse and dissolve, but remain at the introduction site. UCNPs cause an inflammatory reaction in skin, in a week the skin site, where particles were administrated, is almost not seen. The changes in blood supply and blood circulation in the organs were developed in 2 days mainly. This in turn led to insignificant dystrophic changes. All changes were reversible and disappear or their degree of severity decreases after 7 days after the administration of the particles. The particles enter the total blood flow, but they did not show a pronounced toxicity.

Diabetic retinopathy damages retina due to diabetes mellitus which leads to blindness. Here, we have applied local binary pattern (LBP) in order to capture the spatial variations of the refractive indices due to progress of diabetic retinopathy among retinal tissues. After extraction of discriminative textures as binary numbers, state of art machine learning algorithms like decision tree and K-NN have been applied to get the optimum detection accuracy in multiclass classifications of in vivo diabetic retinopathy images. Here it is quite apparent that K-NN provides better accuracy and specificity than decision tree.

In today’s point-of-care testing (POCT), there is an ever-increasing demand for novel and more efficient devices for early diagnosis, especially in cardiovascular diseases (CVD). Early detection of CVD markers, such as Troponin present in the bloodstream, is a key factor for reducing CVD mortality rates.

Thiol-ene coupling (TEC) and Light Assisted Molecular Immobilization (LAMI) are photonic techniques leading to immobilization of bioreceptors, such as, antibodies which recognize cardiac markers. These techniques present advantages compared to traditional immobilization techniques since, e.g., there are no thermal or chemical steps and they work in water media. TEC reaction takes place at close-to-visible wavelengths (λ=365nm) which induces the formation of thiol radicals which bind to alkene functional group on the surface through a thioether bond. LAMI secures molecular immobilizations in a spatially oriented, localized and covalent coupling of biomolecules onto thiol reactive surfaces down to submicrometer spatial resolution. LAMI is possible due to a conserved structural motif in proteins: the spatial proximity between aromatic residues and disulfide bridges. When aromatic residues are excited with UV light (275- 295nm), disulphide bridges are disrupted and free thiol groups are formed that can bind covalently to a surface decorated with thiol groups.

We have achieved successful immobilization of anti-troponin and anti-myoglobin antibodies with both photonic immobilization techniques. The microarrays of immobilized monoclonal antibodies have successfully detected the CVD biomarkers troponin I and myoglobin, as confirmed by fluorescence imaging. A sandwich immunoassay was carried out, Troponin I and Myoglobin were detected down to 10 ng/mL and 1 ng/mL, respectively.

Local hemodynamic parameters were studied by means of laser Doppler flowmetry in 15 patients with psoriasis in the stationary stage, who have plaques on the inner surface of the forearm.

LDF signals recorded at the site of psoriatic lesions of the tissue as well as in the intact tissue at a distance of 1-2 cm from the affected area were analysed. LDF signals were postprocessed by continuous wavelet transform using the Morlet wavelet.

As the incidence of skin cancer is still increasing worldwide, there is a high demand for early, non-invasive and inexpensive skin lesion diagnostics. In this article we describe and combine two skin imaging methods: skin autofluorescence (AF) and multispectral criterion p’. To develop this method, we used custom made prototype with 405 nm, 526 nm, 663 nm and 964 nm LED illuminations, perpendicular positioned linear polarizers, 515 nm filter and IDS camera. Our aim is to develop a skin lesion diagnostic device for primary care physicians who do not have experience in dermatology or skin oncology. In this study we included such common benign lesion groups as seborrheic keratosis, hyperkeratosis, melanocytic nevi and hemangiomas, as well two types of skin cancers: basal cell carcinoma and melanoma. By combining skin AF and multispectral p’ imaging methods, we achieved 100% sensitivity and 100% specificity for distinguishing melanoma (3 histologically confirmed cases) from seborrheic keratosis (13 dermatologically confirmed cases), hyperkeratosis (8 histologically and 1 dermatologically confirmed case), melanocytic nevi (23 dermatologically confirmed cases ), basal cell carcinomas (2 histologically and 16 dermatologically confirmed cases) and hemangiomas (8 dermatologically confirmed cases). Unfortunately, currently this method cannot distinguish the basal cell carcinoma group from benign lesion groups.

Soft biological tissues manifest strongly pronounced nonlinear elastic behavior. Namely, the Young modulus for some tissues strongly depends on the applied stress. This property can be evaluated by compressional OCT elastography. We demonstrate the evaluation of nonlinear elastic properties on the samples of coronary arteries, breast cancer and cornea. The developed technique can be used for further investigation of the nonlinear properties of healthy and pathological tissues.

Diffuse reflectance spectra of bacterial colonies, from hyperspectral images, allowed for a label-free Gram classification into Gram-positive (GP) and Gram-negative (GN) types. Thirty-eight strains belonging to 14 bacterial species typically encountered in urinary tract infections (UTI) were cultivated on chromID CPS Elite translucent chromogenic culture medium to build training and testing sets. Using Support Vector Machine (SVM) supervised learning models, we demonstrated excellent classification rates with a percentage of correctly classified samples as high as 95%. Because determination of discriminant spectral channels is critical both for fundamental reasons to help understand the origin of the discriminant signal and for practical reasons to envision simpler multispectral systems, parsimonious analysis was conducted employing a Fused LASSO (Least Absolute Shrinkage and Selection Operator) or based on an uncertainty test in Partial least squares PLS regression analysis. Two prominent distinct spectral regions were thus identified allowing to hypothesize that cytochrome ratios might be, at least in part, at the origin of the differences observed between Gram-negative and Gram-positive bacteria populations.

Ultrasound-guided diffuse optical tomography (DOT) is very favorable and promising imaging technique for the early detection of breast cancer. The technique uses a hand-held probe capable of providing multiple wavelengths measurements in few seconds. These measurements are used to estimate optical proprieties of lesions inside tissue, and calculate the total hemoglobin concentration (tHb). The accurate recovery of breast lesion optical properties requires an effective image reconstruction method. Errors in the measurements caused by low signal-to-noise ratio data and/or movements during data acquisition would reduce the accuracy of reconstructed optical properties. Here is an attempt to preprocess the data based on multiple wavelength measurements for image reconstruction. This approach combines data collected from multiple sets of lesion measurements of different wavelengths to detect and correct outliers in the perturbation. The initial results obtained from the phantom experiments are promising. The approach has enhanced the consistency of the reconstructed images among multiple wavelengths.

Introduction of innovative noninvasive diagnostic techniques in clinical practice remains an actual topic of modern medicine. In the study, we assessed feasibility of multimodal OCT (MM OCT) that combines crosspolarization imaging and elastographic stiffness mapping to assess spatial structural organization and heterogeneity of breast cancer in the tumor center in comparison with normal mammary gland tissue. The research was carried out using human breast tissue mastectomy surgical samples including different histological types of breast cancer: ductal carcinoma in situ, invasive lobular and ductal carcinoma, fibroadenoma and normal tissue. The histological subtypes of breast cancer showed different structural and stiffness features of tumor tissue. In the case of invasive ductal carcinoma, the cross-polarization OCT image shows a more heterogeneous high-level OCT signal and higher stiffness in comparison with lobular breast cancer or fibroadenoma. These results indicate that assessing microstructures and elasticity changes yields complementary information about the microstructural features of breast cancer, which is important for selection of treatment tactics.

Knowledge of the optical properties of tissues is necessary, since they change from tissue to tissue and can differ between normal and pathological conditions. These properties are used in light transport models with various areas of application. In general, tissues have significantly high scattering coefficient when compared to the absorption coefficient and such difference usually increases with decreasing wavelength. The study of the wavelength dependence of the optical properties has been already made for several animal and human tissues, but extensive research is still needed in this field. Considering that most of the Biophotonics techniques used in research and clinical practice use visible to NIR light, we have estimated the optical properties of colorectal muscle (muscularis propria) between 400 and 1000 nm. The samples used were collected from patients undergoing resection surgery for colorectal carcinoma. The estimated scattering coefficient for colorectal muscle decreases exponentially with wavelength from 122 cm^-1 at 400 nm to 95 ^cm-1 at 650 nm and to 91 cm^-1 at 1000 nm. The absorption coefficient shows a wavelength dependence according to the behavior seen for other tissues, since it decreases from 8 cm^-1 at 400 nm to 2.6 cm^-1 at 650 nm and to 1.3 cm^-1 at 1000 nm. The estimated optical properties differ from the ones that we have previously obtained for normal and pathological colorectal mucosa. The data obtained in this study covers an extended spectral range and it can be used for planning optical clearing treatments for some wavelengths of interest.

In the present work an influence of interactions between detonation nanodiamonds and biomacromolecules (DNA and lysozyme) on fluorescent properties of nanodiamonds was studied. Formation mechanisms of complexes of nanodiamonds with DNA and lysozyme molecules were investigated. It was found that fluorescent intensity of detonation nanodiamonds changes in different ways for nanoparticles with different surface composition. It was established that fluorescent intensity of nanodiamonds increases in case of the interaction with a sufficient number of biomacromolecules.

Optical coherence tomography (OCT) is a promising method of glial tumors borders diagnostics. Nowadays it is possible to use hand-held and microscope mounted OCT devices during tumor removal. But still there are no clearly visual assessment criteria of OCT images, on the basis of which good differentiation between glioma tissue and white matter can be performed. This paper presents such criteria for crosspolarization OCT (CP OCT), which can detect both the scattering and polarization properties of tissues.

The development of photomedical modalities for diagnostics and treatment has created a need for knowledge of the optical properties of the targeted biological tissues. These properties are essential to plan certain procedures, since they determine the light absorption, propagation and penetration in tissues. One way to measure these properties is based on diffuse reflectance spectroscopy (DRS). DRS can provide light absorption and scattering coefficients for each wavelength through a non-invasive, fast and in situ interrogation, and thereby tissue biochemical information. In this study, reflectance measurements of ex vivo mice organs were investigated in a wavelength range between 350 and 1860 nm. To the best of our knowledge, this range is broader than previous studies reported in the literature and is useful to study additional chromophores with absorption in the extended wavelength range. Also, it may provide a more accurate concentration of tissue chromophores when fitting the reflectance spectrum in this extended range. In order to extract these concentrations, optical properties were calculated in a wide spectral range through a fitting routine based on an inverse Monte-Carlo look-up table model. Measurements variability was assessed by calculating the Pearson correlation coefficients between each pair of measured spectra of the same type of organ.

In this work, tissue-engineered structures based on a matrix of protein conjugates, chitosan and carbon nanotubes were prepared and studied. Bovine serum albumin (BSA), bovine collagen (BС) were used. Two types of single-walled carbon nanotubes (SWCNTs) were used to form a strong internal scaffold in a protein-chitosan matrix under the action of laser radiation. Tissue-engineered structures were created by means of layered deposition and laser evaporation of the initial aqueous dispersion from SWCNT, BSA, BC and chitosan succinate. As sources of laser radiation, a continuous diode laser with a wavelength of 810 nm and a pulsed fiber laser with a wavelength of 1064 nm and frequency of 80 kHz were used. Studies of tissue-engineered structures were carried out using vibrational spectroscopy methods (IR and Raman). The changes in the frequencies and intensities of the corresponding absorption bands and Raman lines of the amide group oscillations were analyzed. IR spectra of tissue-engineered structures demonstrated a high degree of binding of organic (protein, chitosan) and inorganic (SWCNT) components. The structure and defectiveness of the carbon nanotube scaffold were investigated in the Raman spectra.

The experimental setup for multispectral nanoparticle tracking analysis is developed. The optical system for sample illumination in nanoparticle tracking analysis is designed that makes it possible to obtain images of particles in suspensions with a high signal-to-noise ratio. The results of the comparison of particle size distributions obtained by nanoparticle tracking analysis using developed experimental setup, dynamic light scattering method and scanning electron microscope are shown.

Nowadays skin pathology detection on early stages is one of the progressive directions in medicine. Optical diagnostics methods allow to obtain data on skin's structure and composition,to examine biotissue processes without any negative impact on skin. Polarization diagnostics methods are very perspective. They are of interest due to biotissue-scattered optical fields polarization characteristics' high sensitivity to the structural features and physiological status of the study object. Implementation of quantitative ellipsometry in turbid media eventually can provide qualitatively new results with studies of the tissue's morphological and functional state which relate to the most important directions of today medical diagnostics. As a result development of theoretical and practical aspects of optically heterogeneous biological structures and polarized radiation interaction while solving the tasks of non-invasive rapid medical objects' state control is considered to be of current interest for improving of skin formation detection quality. The purpose of this study is to develop a stand for in vivo skin state examination by scattering ellipsometry with a help of studying of anisotropic skin environment influence on the polarization state evolution of the radiation propagating in this skin.The possibility of implementation the method of quantitative ellipsometry for in vivo skin's optical anisotropy and structural heterogeneity studies is shown. The Mueller matrix algebra is used for describing polarization properties of the depolarizing opticallyactive biotissue medium. A setup for recording the polarization state of the backscattered radiation was designed based on a comparative analysis of the technical options and their application in experiments with biotissue. To have a uniform intensity distribution along the cross section of the input radiation beam, and also to form the polarization states necessary for the study, using the emitting channel of the LEF-3 ellipsometer in the optical scheme of the stand is proposed. The choice of the radiation source wavelength in the spectral range (He-Ne laser, 632 nm) is justified, in which radiation scattering in turbid biological media predominates over absorption, which makes it possible to tell about the sample structural parameters by changing output radiation polarization state. The receiving channel of the output polarization state analyzer was designed, which contains based on a color matrix sensor with a unified analysis field video information block, that allows further multispectral studying of the skin surface structure. The method of a skin ellipsometric examination based on the distribution visualization of the polarization state parameters along the cross section of the output radiation beam and on its subsequent analysis is proposed. For image processing and calculation of the sample polarization characteristics an algorithm and software are developed with a Python language. In the backscattering mode of probing laser radiation the distributions of the skin sector containing scar structures polarization characteristics are obtained.

Photoacoustic microscopy (PAM) is a biological visualization technique that can provide high spatial resolution and high contrast images of deep structures in living tissues. In PAM, the lateral resolution is determined by the size of the focus spot. Generally, because the wavefront aberration, due to the difference of refractive index between samples and air (water) and the shape of samples, enlarges the focus spot, obtained deep images are blurred or distorted. In order to solve this problem, we corrected the wavefront aberration occurring in samples using a transmissive liquid-crystal adaptive optics (AO) element. Our AO element consists of three liquid-crystal layers which have different ITO (indium tin oxide) patterns and are controlled independently. Their patterns are designed to correct the wavefront aberration suitable for a 40X waterimmersion objective lens. The AO element with transmissive and thin structure is easily installed in the PAM system. Also, our AO element is inexpensive and has low power consumption. In this study, we compared photoacoustic images obtained without and with the AO element for a USAF test target, polystyrene beads diffused in glycerol and various tissue specimens. As a result, we found that the use of transmissive AO element improves the lateral resolution and signal-tonoise ratio in PAM.

The interaction between biological tissues and light of a certain wavelength is influenced by the optical properties of each tissue. These are typically estimated from of the measurements of the main characteristics of the radiations pathway.

We herein present our approach that uses a customized version of the Monte Carlo Multi-Layer Algorithm (MCML)¹ to simulate the radiation propagation through biological tissues. We assumed a set of optical properties for each tissue and simulated the above mentioned measurements in silico. A comparison was then done between the results of the simulation and the results of real measurements.² Further, an optimization algorithm searched the set of optical properties that best fit the real optical properties of each tissue. This algorithm was based on adaptions of the Monte-Carlo Simulated Annealing algorithm³ and the Downhill Simplex algorithm⁴ We implemented the MCML using NVidias CUDA application programming interface to speed up the optimization procedure. We validated the software by using van de Hulsts table for Henyey-Greenstein scattering.² A linear regression resulted in coefficients of determination between 0.929 and 0.973 for the optical properties. Our results prove that our algorithm can be effectively used for the determination of the optical properties of turbid media.

One first application for this software is the support in the development of a new generation of hearing devices based on optical energy.

The conductivity of layers (thickness ~ 0.5-20 μm) of composite nanomaterials consisting of bovine serum albumin (BSA) with single-walled carbon nanotubes (SWCNTs) has been studied. The BSA/SWCNT composite nanomaterial was prepared according to a route map, some steps of which are: the preparation of an aqueous dispersion based on BSA and SWCNT; preparation of substrates; deposition of BSA/SWCNT dispersion on substrates; application of water paste from SWCNT on substrates; irradiation of layers by lasers when they were in a liquid state; drying of samples; carrying out electrical and temperature measurements. Half of the layer was covered with a light-tight hollow box and the other half of the layer was laser irradiated. The laser irradiation of the layer was carried out for about 20 sec, at which time the layers completely became dry, while the other half of the layer remained in liquid. Conductivity was increased (70 ÷ 650) % by laser irradiation of the layers when they were in the liquid state. Maximum values of specific conductivity for BSA/SWCNT-1 S/m layers, and for layers SWCNT - 70 kS/m. The investigated electrically conductive layers of 99 wt.% BSA/0.3 wt.% SWCNT are promising for medical practice.

A novel markerless near infrared (NIR) laser-based head tracking system was recently proposed to resolve patient's head motion problem during cranial radiotherapy. Although previous research showed that we could track the patient's head position with the sub-millimetre range accuracy, the tracking performance strongly relied on the accuracy of the tissue thickness estimation. We noticed the factors that inuence the ROI features extracted from the backscattering images were the NIR laser power fluctuation and inconsistency. Therefore, the propose of this paper was to investigate the relationship between these parameters and determine a laser power independent feature transformation. We set up our head tracking system to project the pulsed NIR laser beam onto a single point on subject's forehead and observed the changes of the 5-ROI feature values on the different laser power level. The scatter plots between each ROI feature values and the laser power showed distinctive straight lines with similar slopes while applying linear regression to each scatter plot indicated that the slope of each ROI feature was also in the same range. According to the results, we could transform the data by subtracting the feature value of each ROI from their average slope value and the laser power. This new feature is laser power noise tolerance and could be used to enhance the tissue thickness estimation accuracy.

Optical spectroscopic measurements with a few different modalities have been performed namely autofluorescence, transmission and diffuse reflectance spectroscopies in ultraviolet, visible and near-infrared spectral ranges.

The investigated samples were cutaneous tumours ex vivo, obtained after surgical removal and kept in a formalin solution and histological sections from biopsy tissue samples, which were routinely processed for histological analysis. Comparative spectral data for benign, dysplastic nevi and pigmented malignant melanoma lesions, as well as for nonmelanoma skin tumour – basal cell carcinoma, squamous cell carcinoma and benign non-melanin pigmented pathologies – heamangioma and seboreic veruca are presented in the current report.

Fluorescence spectra obtained reveal statistically significant differences between the different benign, dysplastic and malignant lesions by the level of emission intensity, as well by spectral shape, which are fingerprints applicable for differentiation algorithms. In reflectance and absorption modes the most significant differences are related to the influence of skin pigments – melanin and hemoglobin, less pronounced is the influence of structural proteins, such as collagen and keratin. Transmission spectroscopy mode gives complementary optical properties information about the tissue samples investigated to that one of reflectance and absorption spectroscopy.

A reliable connection of dissected biological tissues is a popular problem in modern surgery. In the last decade, two methods of biological tissues connection using laser radiation have been actively developed: laser-assisted vascular repair (LAVR) and anastomosis (LAVA). These methods make it possible to obtain a weld impenetrable for blood and other biological fluids immediately after the welding. A solder is applied to a welding area. The main characteristic of the weld at LAVA is the tensile strength. A weld should be flexible enough to withstand repeated cycles of alternation of diastolic and systolic pressures. Single-walled and multi-walled carbon nanotubes (SWCNTs and MWCNTs respectively) are used to increase the weld tensile strength. They form a spatial bovine serum albumin (BSA)- nanocarbon scaffold under an action of laser radiation. It in real time varies the power of laser radiation (in the range 0.2- 5 W), which is necessary to stabilize the welding temperature (~57 ºC). In the present work new compositions of laser solder are proposed and the scheme of the installation for LAVR are offered. The proposed solder is based on an aqueous dispersion of BSA, MWCNTs and SWCNTs, ICG and collagen. The using of the chromophore ICG is due to its absorption maximum corresponding to the wavelength of the diode laser used (~ 810 nm). The tensile strength was 0.8±0.3 MPa.

Three-dimensional (3D) polymeric scaffolds are utilized in tissue engineering to provide biomechanical support for the seeded cells until they are organized into a functional tissue. Studies of cell migration are important for understanding a variety of physiological and pathological processes in tissue regeneration. Optical techniques can be employed for monitoring cell migration processes in 3D scaffolds. In particular, two-photon microscopy (TPM) can be used for monitoring these processes for living cells also at considerable depths. In the present work, the cell migration process of alive chondrocytes labelled with vital PKH67 in 3D collagen-based scaffold has been monitored at different times from the seeding process. The results show that TPM can evidence the presence of living cells in different spatial regions whose localizations depend on the location and time elapsed from the seeding.

The present study introduces recently developed compact hyperspectral snapshot system (device and software) for skin oxygen saturation monitoring. This prototype device involves compact snapshot hyperspectral camera, multi-wavelength illuminator, optical filter and crossed polarizers. The device was validated using reference color samples and and in-vivo during finger arterial occlusion tests. The prototype system demonstrated good performance of skin hyperspectral measurements in spectral range of 500-630nm. The results confirmed reliability of developed system for in-vivo assessment of skin blood oxygen saturation.

One of the most relevant postoperative problems in liver transplantation is the initial graft dysfunction, which is generated by the oxidative stress due to ischemia and/or reperfusion. This leads to ischemia-reperfusion injury of the transplanted organ. This injury can be severe and, when the primary graft is nonfunctional, an urgent retransplantation is required. In this context, tools to monitor the oxidative stress in liver grafts would improve the surgical decision-making for transplantation, increasing its success rate. In this study, we evaluated the potential of time-resolved fluorescence spectroscopy to measure oxidative stress in liver grafts before transplantation. This was performed in livers after ischemia and reperfusion kept in 0 °C (control group) and or 20 °C (ischemia-reperfusion injury group). Both fluorescence spectra and lifetimes were monitored immediately, each 1 hour for the first 12 hours, and at 24 hours after the removal of the liver. The hepatic tissue was excited by lasers emitting in 378 nm and 445 nm for investigation of possible metabolic rates associated to NAD(P)H, FAD, lipopigments, and lipofuscin molecules. The fluorescence decay curves were fitted to the convolution between the instrument response function with a bi-exponential decay for 378 nm and a tri-exponential decay for 445 nm by using the SPCImage software. For both excitation wavelengths, the relative weights for the first exponential component decreased faster as a function of time for the ischemia-reperfusion injury group compared to the control group. This suggests time-resolved spectroscopy is a promising technique to help clinicians to make decisions before liver transplantation.

In this study, the optical properties of glycated (HbA1c) and non-glycated (Hb) hemoglobin are compared using refractometry, fluorescence and Raman spectroscopy. The fluorescence measured at an excitation wavelength of 270 nm indicates differences in the molecular structure of hemoglobins. Analysis of the spectral shift of Raman spectra also showed variations indicating differences in their molecular structure. The refractive index measured in the visible and near IR regions for different temperatures allowed for quantification of mean values of temperature increment, which are quite different as dn/dT= –(1.03 ± 0.05)×10^–4 °C^–1 for Hb and – (1.37 ± 0.07)×10^–4 °C^–1for HbA1c.The data obtained in the work can serve as a basis for further study of the optical properties of glycated hemoglobin and other glycated proteins.

Multispectral diffuse reflectance imaging and autofluorescence photo-bleaching imaging are methods that have been investigated for use in skin disorder diagnostics. In response to the ever-increasing incidence of skin cancer in light skinned populations a new device has been designed incorporating both of these methods. The aim of the study was to create a device that is most efficient in terms of hardware and software parameters for the screening of malignant and benign skin lesions. A set of 525 nm, 630 nm and 980 nm LEDs were used to illuminate the skin area at three wavelengths [1] and a set of 405 nm LEDs were used to induce the skin autofluorescence [2]. For a more homogenous illumination of investigated skin area the optimal placement for LEDs in a cylindrical case was found. The requisite spacing from the camera lens was taken into account to produce a focused RGB image. The geometrical shape of the device allows to capture images of skin that are illuminated solely by the diodes without interference from sunlight or other nearby light sources. Polarizing filters were used to decrease glare effects, therefore preventing image overexposure of very reflective skin areas. 515 nm long pass filter was used to enable the 405 nm excitation while capturing autofluorescence images of the skin. Further improvements to the quality of the diagnostic data can be achieved using reference images to track homogeneity of the intensity and then applying a compensating algorithm on the subsequent screening images. These and other design considerations serve to realize the full potential of the diagnostic method.

Results of clinical approbation to assess the efficacy of the new device to diagnose malignant skin lesions will be demonstrated.

It is a well-known fact that the raw photoplethysmography signal contains a slowly variable respiratory component which can be extracted by a low-frequency filtration or spectral decomposition. However, these methods do not allow to determine the current phase of the respiratory cycle, i.e. inhalation, exhalation, or plateau phase. And yet these often subtle differences in the characteristics of the individual phases of breath cycle and their interactions have a clinically significant diagnostic value. During the work, it was noted that the chest movements during respiratory cycle contribute to the shape of the PPG curve – especially on the descending part of the photoplethysmographic waveform. This effect was used to develop a method for evaluating a phase of respiratory cycle on-the-fly. The proposed method is based on the temporal variation of the three characteristic points (tPA, tPB and tPC) of the normalized PPG curve. The tPA designator is the reference point for an algorithm that normalises the amplitude of the PPG signal. Points tPB and tPC (in particular their variability over time) are the basis for further analysis that leads to extraction of a phase of respiratory cycle. Strong correlation between the respiratory curve and the temporal variation of the tPB and tPC points allows to assess the respiratory cycle phase on-the-fly.

Long exposure to ultraviolet A, UVA (315 nm - 400 nm) and ultraviolet B, UVB (280 nm - 315 nm) from the sunlight is harmful to human skin. It can cause unwanted pigmentation, skin aging, wrinkles and cancer. Thus, application of sunscreen lotion is necessary to protect us from these harmful ultraviolet. However, sunscreen lotion does not last for an entire day, its effectiveness degrades over time. In this study, we aim to investigate the effectiveness of various types of sunscreen lotion. The ingredient of the chosen sunscreen includes common ingredient such as ethylhexyl methoxycinnamate and titanium dioxide. In this experiment, ethylhexyl methoxycinnamate show degradation after exposed to tungsten-halogen lamp for 180 minutes. However, mixing titanium dioxide into ethylhexyl methoxycinnamate did improve its stability and protection against UVA and UVB.

Visible light communication (VLC) has various advantages over radio frequency (RF) communication such as ubiquity, low energy consumption, no RF radiation, and inherently secure as light does not penetrate through walls. Significant bio-medical signals including the electroencephalography (EEG) can be transferred with VLC even in places where RF is forbidden. Additionally, long-term exposure to RF radiation poses a risk to the human brain which limits the use of RF wireless wearable EEG systems. This potential advantage of VLC could help in the indoor healthcare system such as monitoring. A long-term video-EEG monitoring requires continuous monitoring by video along-with EEG signals. So, overall a significant amount of data needs to be streamed fast for real-time monitoring. A novel low-cost RF radiation-free system is proposed using VLC technology which can be integrated into a wearable EEG device. In this work, we transmit a video and multi-channel EEG signal using visible light communication. Data streams are modulated using colour shift keying (CSK) which drives the RGB LED. CSK gives double the data rate than OOK by mapping bits into a symbol. It keeps the average emitted optical colour constant during communication, thereby reduces potential human health complication related to light ashes. The proposed system aims to lower down the cost and complexity further by using single photodiode at the receiver unlike conventional CSK in IEEE 802.15.7. The receiver architecture exploits channel estimation in a better way. Computer simulations are carried out using actual raw EEG signals and video data. Simulated and theoretical SER versus SNR curves match seamlessly. The results demonstrate that at 33 dB SNR, it achieves a SER of 1x10^-5. SER for different transmitter and receiver distance is also analysed. Further, the reliability and accuracy of data received at 33 dB is also discussed.

Monitoring blood-brain barrier (BBB) opening is of great interest in terms of brain drug delivery in the treatment of brain lymphoma and maybe in the future in other diseases like dementia. A method involving BBB disruption (BBBD) by mannitol infusion has been developed in University of Portland, USA, and then exploited in Oulu University Hospital in treatment of primary CSN lymphoma. Proper opening of the BBB is crucial for the treatment, yet there are no methods available for its real-time clinical monitoring. Recently, we presented a combined method using direct-current electroencephalography (DC-EEG) and near-infrared spectroscopy (NIRS) for monitoring BBBD in human. Carotid artery mannitol infusion generated a strongly lateralized DC-EEG response and in NIRS a prolonged increase in the oxy/deoxyhemoglobin ratio.

This study explores further BBBD, by focusing on monitoring its cardiovascular effects, when measured in human and mouse. For this, we used photoplethysmography (PPG) and opto-electro-mechanical sensors to gather the signals in human and mouse. Mannitol infusion in human causes strong fluctuations in blood pressure, heart rate and PPG signals, and here we discuss how the acquired signals in mouse model compares to human data.

In addition, we present our scale-free monitoring concept that enables monitoring physiological signals similarly when performing experiments in mouse and human neuroimaging setups. By combining microscopic and macroscopic imaging in mouse setup enables us to study correlations between mechanistic cellular data and clinical functional data. Further, this allows us to validate and optimize macroscopic sensing and imaging techniques aimed to be used in human imaging.

Laser Doppler flowmetry (LDF) was utilized for blood ow measurements. Wavelet analysis was used to identify spectral characteristics of the LDF signal in patients with rheumatic diseases and diabetes mellitus. Baseline measurements were applied for both pathological groups. Blood flow oscillations analyses were performed by means of the wavelet transform.

Higher baseline perfusion was observed in both pathological groups in comparison to controls. Differences in the spectral properties between the groups studied were revealed. The results obtained demonstrated that spectral properties of the LDF signal collected in basal conditions may be the signature of microvasculature functional state.

The aim of the presented work is to investigate the angle-resolved scattering characteristics of biological nano- and micro-scaled cell structures. The scattering results of cellular structures were compared to measurements of ideal spherical nano- and micro-particles. A monolayer of mouse fibroblasts L929 cells was cultivated in a Dulbecco's Modified Eagle Medium (DMEM) in a standard 24 well cell culture plate. The system allows an in situ measurement directly in the standard cell culture plate and a contaminant-free investigation of the viability of the cell cultures. Of particular interest was whether changes in the tumor characteristics occur in apoptosis or other cell-harming effects. Because of the size ratios between wavelength and the scattering particles, all observations were investigated using Mie scattering theory. A setup for reliable measurements was developed and the scattered angle dependent intensity obtained was compared with simulated scattering characteristics. A homemade supercontinuum (SC) light source was filtered by an optical bandpass filter with a central wavelength of 500 nm. The scattered portion of the pulsed SC light behind the sample was recorded in a time-resolved manner at defined angles. A specimen holder adapted to standard cell culture plates allows detection of scattered radiation at angles between ±80° without angle-dependent Fresnel reflection losses and a Snell’s law bending of the propagation direction. Finally, the system was tested to detect structural changes of mouse fibroblast L929 cells before and after poisoning the cells with the cell detergent Triton X100 and the data clearly shows changes in the scattering characteristics when the cells were destroyed.

The overall aim of this study was verification of the possibility to register the change of NADH fluorescence in live tissue by a portable diagnostical laser system with fibre optical probe output and excitation by compact semiconductor UV light source. The measurements were conducted in fresh brain tissue slices of Wistar rat pups. The fluorescence measurements were conducted simultaneously at intervals of 5 s by the microscopic system with excitation at 360 nm and registering of the emitted fluorescence light at 455 nm and by the tested diagnostical system equipped with the fibre optical probe with excitation at 365 nm and registration of the fluorescence spectrum by the inbuilt spectroscopic subsystem. To modulate the mitochondrial function in the living cells, in the chamber sequentially were added 1 μM FCCP and 1 mM NaCN. The comparisons between the curves registered by the methods allowed us to find well agreement between the microscopic measurements and measurements by the fibre optical probe. The obtained results prove that the tested diagnostic system is capable of sensing the changes in brain metabolic activity associated with the NADH content alterations within the physiological range.

Raman microspectroscopy is an optoelectronic technique that can be used to evaluate the chemical composition of bio- logical samples. Raman spectroscopy has been shown to be a powerful classification tool for the investigation of various cancer related diseases including bladder, breast and cervical cancer. Raman scattering is an inherently weak process with approximately 1 in 10⁷ photons undergoing scattering. For this reason, noise from the recording system can have a significant impact on the quality of the signal and its suitability for classification. Different camera settings when obtain- ing spectra from charge-coupled devices can result in significantly different noise performance. This paper provides an investigation into practical aspects of retrieving the signal from the charge-coupled device, and the effects of integration time and multiple acquisitions on the signal to noise ratio of Raman spectra, with a particular focus on biological sam- ples. The main sources of noise are shot noise, CCD dark current, readout noise, and cosmic ray artefacts. Shot noise and dark current noise are time dependent and so there are practical considerations when choosing an integration time. Readout noise is inherent in each individual recording, which may be compounded when averaging spectra together. Our results demonstrate that read parameters and read modes can greatly influence the signal to noise ratio. We also discuss experimental conditions and processing methods that can mitigate these effects.

Non-linear optical imaging techniques have been used to greatly enhance our understanding of issues with high biological significance and promise a strong impact on early and accurate detection of various diseases. In our current work, we employ Third Harmonic Generation (THG) and Second Harmonic Generation (SHG) imaging modalities for diagnosis of cancerous tissue limits and for obtaining additional quantitative information to supplement standard histopathology procedures. For this reason, unstained histological slides of breast tumor biopsies were irradiated. Cancerous and normal tissue areas could be distinguished based on cell morphology, size, and density. THG imaging reflects lipid bodies (LBs) in the intracellular compartments, cellular and nuclear membranes, while SHG shows collagen distribution in the tissue. By using THG microscopy, it is feasible to concentrate on specific cells in tissue and collect quantitative information. Our initial results showed that quantification of THG signaling can depict differences between healthy and cancerous tissue. This is a very promising observation, since the non-linear technique described here, allows fast, non-invasive, label-free imaging that does not require the use of fluorescent dyes or other preparations of tissues in order to detect specific structures and features. The significance of this work has a clinical potential since it can monitor quantitative changes in cellular behavior in healthy and pathological human tissues.

In this work, we employed the Raman microscopy to study the internalization kinetics and spatial distribution of small interfering RNA (siRNA)-diatomite nanoparticles (DNPs) complex in human lung epidermoid carcinoma cell line (H1355) up to 72 h. Raman images are compared with confocal fluorescence microscopy results. The Raman analysis provides that the siRNA-DNPs are internalized and co-localized in lipid vesicles within 18 h, after that equilibrium is achieved.

Optical properties of flavin adenine dinucleotide (FAD) moiety are nowadays widely used for biotechnological applications. Given the fundamental role played by FAD, having additional information about this complex can be extremely useful to get a deeper insight in structural properties and functional role of this enzymatic co-factor. For this purpose, we investigated FAD behaviour in aqueous solutions at two pH values by means of complementary optical spectroscopic techniques (circular dichroism, infrared spectroscopy and time-resolved fluorescence). The results confirm that pH influences circular dichroism spectra. An innovative Maximum Entropy Method was adopted for time-resolved fluorescence signal analysis allowing us to evidence a three-components decay for FAD in aqueous solution with pH-depending lifetimes and relative amplitudes. All the results here reported give a more complete view of FAD’s properties that can be exploited in designing new biotechnological devices.

Laser Doppler flowmetry (LDF) is widely used to study blood microcirculation in the skin. However, during tradition signal processing based on the integral estimations of the power spectrum of detector photocurrent, the significant part of the information about the skin blood ow is lost. In this study, we propose to analyse the distribution of the blood perfusion over the Doppler shift frequencies, which correlate with the RBC velocity. This approach provides localisation of the blood ow oscillations in different subranges of the Doppler shift. The method applied together with the wavelet analysis has been tested in healthy volunteers and patients with psoriasis on the unaffected surface of the skin. It was revealed, that the significant difference in the amplitude of myogenic oscillations is allocated in the region of the low frequency Doppler shift (1-200 Hz). This frequency region can be associated with the signal from slow components of the skin microcirculation, that can point out on a different state of the lymphatic system of the skin in psoriasis.

At present, minimally invasive interventions become more widespread for treating hepatopancreatoduodenal area pathologies. However, new methods and approaches are necessary for obtaining more diagnostic information in real time. Several methods within the framework of “optical biopsy” concept are considered. The features and areas of application of each method are reviewed to find out which of them can be used in further studies to assess the possibility of intraoperative use in minimally invasive abdominal surgery. Preliminary measurements with fluorescence spectroscopy method have been performed at excitation wavelengths 365 nm and 450 nm. Areas of interest were common bile duct, gallbladder and liver abscess. In our opinion, the obtained results can be a basis for further research and provide a deeper understanding of pathological processes of abdominal cavity organs tissues.

This studiy was carried out on groups of clinically healthy male Wistar rats. Animals received distilled drinking water ad libitum for 1 month, water containing succinic acid, water containing zinc sulphate and succinate zinc. Using the method of fluorescence spectroscopy, the parameters of brain metabolism in vivo in a model of laboratory rats was investigated. Based on data obtained by fluorescence spectroscopy, we have registered a change in the degree of cellular respiration in different structures of the cerebral cortex with the toxic effect of zinc compounds and succinic acid on the oxygen exchange process.

Laser Doppler flowmetry (LDF), tissue reflectance oximetry (TRO) and pulse oximetry (PO) and cold pressor test (CPT) were used to assess the microcirculation parameters and the activation of regulatory mechanisms. LDF and TRO samples wavelet transform in the frequency bands 0.01-2 Hz was used to evaluate microvascular disturbances in rheumatic diseases and to assess the vascular involvement in the pathological process. The spectral components of LDF and TRO signals associated with endothelial, adrenergic, intrinsic smooth muscle, respiratory and cardiac activities were analyzed. Significant difference between healthy and rheumatology subjects was identified in perfusion parameters. Spectral analysis of the LDF signal revealed significant difference between two group of high (<0.1 Hz) frequency pulsations. Based on the analysed of the perfusion and amplitudes oscillation in the frequency band the decision rule for detection microvascular disturbances were synthesized. The perfusion parameter and amplitude oscillation associated with cardiac activities included in the decision rule. Based on the measured parameters and the result of wavelet transform LDF- and TRO-signals the parameters for detection of complications associated with microvascular disturbances and their possible causes were proposed..

The goal of this study was to assess the ability of singlet oxygen generated at photodynamic treatment using different irradiation modes to inhibit the enzyme TAQ polymerase activity. Experimentally were determined the laser irradiation dose and the singlet oxygen concentration that results to complete inhibition of TAQ polymerase activity. The results show that using pulse mode irradiation is 1.5x more efficient then continuous wave. TAQ polymerase damage was also assessed by fluorescence spectrometry — tryptophan residues fluorescence is decreased if damaged by singlet oxygen during photodynamic treatment. It was observed that on doses, where TAQ polymerase looses its enzymatic activity the fluorescence decreases only by 7%. The fluorescence decrement of tryptophan correlates with damage to the enzyme.