This PDF file contains the front matter associated with SPIE Proceedings Volume 11075, including the Title page, Copyright Information, Table of Contents, Author and Conference Committee lists.

Direct and continuous measurements of blood flow are of significant interest in many medical specialties. In cardiology, intravascular physiological measurements can be of critical importance to determine whether coronary stenting should be performed. Intravascular pressure is a physiological parameter that is frequently measured in clinical practice. An increasing body of evidence suggests that direct measurements of blood flow, as additional physiological parameters, could improve decision making. In this study, we developed a novel fibre optic intravascular flow sensor, which enabled time-of-flight measurements by upstream thermal tagging of blood. This flow sensor comprised a temperature sensitive polymer dome at the distal end of a single mode optical fibre. The dome was continuously interrogated by low coherence interferometry to measure thermally-induced length changes with nanometre-scale resolution. Flow measurements were performed by delivering heat upstream from the sensor with a separate optical fibre, and monitoring the temperature downstream at the dome with a sample rate of 50 Hz. A fabricated flow sensor was characterized and tested within a benchtop phantom, which comprised vessels with lumen diameters that ranged from 2.5 to 5 mm. Water was used as a blood mimicking fluid. For each vessel diameter, a pump provided constant volumetric flow at rates in the range of 5 to 200 ml/min. This range was chosen to represent flow rates encountered in healthy human vessels. Laser light pulses with a wavelength of 1470 nm and durations of 0.4 s were used to perform upstream thermal tagging. These pulses resulted in downstream temperature profiles that varied with the volumetric flow rate.

In recent pre-clinical studies, we have demonstrated that analysis of speckle statistics in low-scattering, low SNR regions may have applications for accurate lymphatic vessels (and nerves) mapping in optical coherence tomography (OCT) of biotissues. Specifically, we utilized a Rayleigh fitting goodness-of-fit threshold parameter to isolate low scattering biological structures, in particular to distinguish them from noise. The threshold as well as the size of region of interest (ROI) for such speckle statistics evaluation were both chosen empirically. Here we present a model simulation study aimed at optimizing and automatically determining these empirically chosen important parameters. We explore the Rayleigh fit and speckle contrast metrics parameter space to enable accurate label-free OCT mapping of weakly scattering structures such as lymphatic vessels.

Skin-remitted picosecond laser pulses have been detected at variable input-output fiber distances (8 … 20 mm) in the spectral range 520-800 nm, with subsequent analysis of the pulse shape changes. Transfer functions representing the temporal responses of remitted photons to infinitely narrow δ-pulse excitation have been calculated. Parameters related to the photon path length in skin – input-output pulse peak delays, pulse FWHM, travel times of the “initial” photons and distributions of the remitted photon path lengths – are presented and analyzed. The measurement results are in general agreement with the photon propagation model expectations

In this study, we present facile fabrication of a miniaturized remote sensing SERS platform using highly tunable Nano- Sphere Lithography (NSL) technique. Self-assembly at the air-water interface was performed and the monolayer of polystyrene spheres was transferred onto the tip of optical fibers. Various optical fibers with different numerical apertures (NAs) were used to find an optimal remote sensing setup. Using 200μm diameter optical fibers with high numerical aperture (0.5NA), the SERS enhancement of remote sensing was found to be 98% of direct sensing configuration. Standard silica optical fibers were used for remote sensing using SERS without additional need of optical filtering to mitigate fluorescence and Raman background of these fibers which allows fabrication of miniaturized remote sensing platforms that can be used for remote biochemical sensing.

Laser Doppler flowmetry (LDF) was used for detection of age-related changes in the blood microcirculation. The LDF signal was simultaneously recorded from the 3rd fingers' pads of both hands. Amplitudes of the blood flow oscillations and wavelet coherence of the signals were used for the data analysis. A statistical difference in the synchronisation of myogenic oscillations was found between the two studied age groups. Myogenic oscillations of blood perfusion in the younger group had a higher wavelet coherence parameter than in the older group. Observed site-specific and age-related differences in blood perfusion can be used in the future in the design of experimental studies of the blood microcirculation system in patients with different pathologies.

Multimodal optical coherence tomography (OCT) techniques are promising diagnostic tools to accurately assess highrisk atherosclerotic plaques. For rapid translation into clinical practice, the techniques should be performed through an intravascular imaging catheter without exogenous contrast agents under the same procedures as conventional imaging. In this study, we developed a label-free, multispectral, and catheter-based imaging system to simultaneously visualize the morphological and compositional information of coronary plaques by combining fluorescence lifetime imaging (FLIm) and OCT. Using a broadband hybrid optical rotary joint and a dual-modal imaging catheter, intravascular combined FLIm-OCT imaging was safely performed in an in vivo atherosclerotic coronary artery of atherosclerotic swine models without any imaging agent. Along with detailed coronary microstructure by OCT, the multispectral FLIm could accurately visualize fluorescence lifetime signature of key biochemical components of plaque in vivo (lipid, macrophage, and fibrous tissue) when comparing the corresponding histopathological stained-sections and ex vivo FLIm microscopy images. Especially, significant differences in fluorescence lifetime distribution were noted between lipid and macrophage (p < 0.0001), which were mostly indistinguishable with standalone OCT. Also, fluorescence lifetime distributions were significantly different according to plaque types (normal, fibrous vs. lipid-rich inflamed plaque, (p < 0.0001). With these statistical differences in plaque types and components, lipid distribution characterization and inflammation level estimation were provided in a pixel-by-pixel manner for the further assessment of the high-risk atherosclerotic plaque. This highly translatable imaging strategy can offer new opportunity for clinical intracoronary detection of high-risk plaques and will be a promising next-generation multimodal OCT technique.

An enhancement of contrast between healthy and neoplastic zones in Mueller matrix images of excised cervical tissue was demonstrated in our prior work for the visible wavelength range. In this paper we present the statistical analysis of Mueller polarimetric data for the diagnostics of high grade cervical intraepithelial neoplasia. The results of linear and non-linear post-processing compressions of the full Mueller matrix are discussed and compared in terms of diagnostic performance. The final goal of these studies is to estimate and compare the diagnostic usefulness of 16 polarimetric measurements required for the reconstruction of complete Mueller matrix of a sample, while looking for an optimal design of future imaging protocols.

Very wide field of view imaging can be used in biology to infer statistical information on cell populations from a singleshot acquisition. In particular, for applications in hematology, fluorescence wide-field of view imaging could be an alternative to standard fluorescence flow cytometry methods; it can be useful to achieve standard blood tests, such a leukocyte count or leukocyte differential count. We will introduce two 30mm² wide-field fluorescence imaging set-ups and compare their performances. Both systems achieve 1x magnification. The first one is based on a macro-photography lens. Although such a system optimizes the resolution and field of view, it suffers from its bulkiness. With the second system, we seek miniaturization while loosening the requirements on image quality. It is based on a lens-less approach with a fiber plate optical relay. The potential of the two systems for hematology analyzes will be illustrated with the imaging of labelled white blood cells.

We developed a smartphone-based epifluorescence microscope for fresh tissue imaging. The smartphone microscope optics was optimally designed to achieve similar resolution (0.56 μm) and FOV (520 μm) as the bench 40x microscope, commonly used during the histopathologic analysis. Preliminary images obtained from an excised human pancreatic tissue stained with a rapid staining fluorescence dye (PARPi-FL) clearly visualized individual tumor cells.

Microscopic whispering gallery mode lasers detect minute changes in cellular refractive index inside individual cardiac cells and in live zebrafish. We show that these signals encode cardiac contractility that can be used for intravital sensing.

Optical tweezers (OT) is a unique Nobel Prize winning technology that has been widely used in studying cell interaction dynamics at a single-cell level with highly accurate manipulating of living cells and the ability to detect ultra-low intercellular forces. The reversible aggregation process of red blood cells (RBCs) that strongly influences the blood rheological properties thus being critical for blood microcirculation has long been research studied. However, in spite of plentiful researches dealing with RBC aggregation behavior based on an average response of a large number of cells, the detailed mechanism and the applicability of the two coexistent yet mutually opposed models to this reversible process require additional information. In this study, a two-channel optical tweezers system is utilized to reveal the influence of the cell interaction time (0-300 sec) on the RBC (dis)aggregation dynamics. The results show that for RBC enforced disaggregation in autologous plasma, the longer the two RBCs adhere to each other, the stronger the intercellular interaction is, and the lower the degree that a certain optical pulling force can separate two cells, whereas no significant effect of cell interaction time on RBC aggregation process was observed. This observation indicates that the RBC aggregation and disaggregation in autologous plasma are governed by different mechanisms and that the hysteresis effect, namely the dependence of the disaggregation force on RBC interaction history, is a significant feature that needs special consideration in researches related to the RBC disaggregation process.

Using attenuated total reflection (ATR) in the terahertz domain, we demonstrate non-invasive, non-staining real time measurements of cytoplasm leakage during permeabilization of live epithelial cells by saponin, and after electropermeabilization. The origin of the contrast observed between cells and culture medium is addressed by both experimental and theoretical approaches, and gives access to permeabilization dynamics of live cells in real time. We show that terahertz modalities are more sensitive than fluorescence microscopy which is the reference optical technique for electropermeabilization. We propose analytical models for the influx and efflux of non-permeant molecules through permeabilized cell membranes.

As a promising drug delivery system, itself or coupled with red blood cells (RBC), nanoparticles (NP) should be studied in frames of their interaction at the cellular level. Experiments were performed on RBC in autologous blood plasma incubated with different NP – TiO2, ZnO, nanodiamonds and polymeric nanocapsules. RBC aggregates formation in RBC suspension was observed with conventional microscopy, while quantitative interaction force measurements between individual RBC was assessed with optical tweezers. Scanning electron microscopy (SEM) imaging demonstrated NP localization and RBC membrane modifications upon binding with NP. Among tested NP, nanodiamonds caused increasing the size of aggregates in RBC suspensions, RBC interaction force increase and strong membrane surface modifications, comparing to other tested NP and control sample. Nanocapsules do not cause any adverse effects on RBC properties, confirming biocompatibility and applicability for drug delivery purposes. Optical tweezers combined with SEM imaging serves as fast informative assessment of NP effects on RBC.

Biocompatible hydrogels present interesting opportunities for in vivo waveguiding for optogenetic or photomedical applications. Here, we investigate the applicability of poly(ethylene glycol) diacrylate hydrogels in combination with scattering particles as optical diffusors. Gel characteristics and bioactivity can be tuned to achieve controlled light distribution and tissue interaction

Spontaneous Raman scattering is a reliable technique for fast identification of single bacterial cells, when spectra are acquired in laboratory conditions where bacteria growth and state are controlled. We have developed a multi-modal system combining Raman spectroscopy and darkfield imaging, aiming at analysing environmental samples, typically in the field context of biological pathogens detection. Such samples are heterogeneous, both in terms of phenotype content and environmental matrix, even after a preliminary purification step. In this paper, we report a study on the identification of Bacillus Thuriengensis (BT) mimicing pathogen bacteria, embedded in a real-world matrix: a sample of surface water enriched with environmental bacterial species. The purpose is to evaluate both the detection limit of aging BT over time and the false alarm rate, in the conditions of our experiment.

Spectroscopic studies of Delayed Luminescence emitted by an in vitro model for studying of the effects of amyloid-βeta (Aβ) have been performed. Aβ is a neurotoxic protein overexpressed in Alzheimer's Disease (AD), which is also related to mitochondrial dysfunction. The experiments have been carried out on primary Olfactory Ensheathing Cells (OECs) cultures. Cells have been exposed to Aβ(1-42) native full-length peptide or to Aβ(25-35), a toxic fragment of Aβ, or Aβ(35-25), a non toxic Aβ fragment, both in absence and in presence of Astaxanthin, a well-known antioxidant agent. To monitor cell viability, MTT test was used. Reactive oxygen species and reduced glutathione levels were utilized to test the oxidative intracellular status. We also assessed the expression of some glial markers (Glial Acidic Fibrillary Protein, Vimentin), of Nestin, stem cell marker, and the activation of the apoptotic pathway assessing caspase-3 cleavage. We found that, in OECs, Glial Acidic Fibrillary Protein, Vimentin expression and caspase-3 exhibited a significant enhancement in Aβ(1–42) and Aβ(25–35) exposed cells. The pre-treatment with Astaxanthin restored the levels of Vimentin and caspase-3 to control values, increasing also Nestin expression levels and reestablished the intracellular oxidative status modified by the exposure to Aβ(1–42) or Aβ(25–35) of OECs. DL intensity and kinetics changes as a function of the treatments were also measured. In particular, an increase in DL emission, with respect the untreated cells (controls), was observed in cells exposed to Aβ(25-35) fragment. This emission appeared quenched in presence of Astaxanthin.

The extra-cellular matrix (ECM) of the tissue significantly remodels during both development and disease. This is often perceived by the apparent changes to the bulk stiffness of the tissue. Nevertheless, the ECM micromechanical alterations and their implications to normal development and pathogenies remain poorly understood, largely due to the absence of high-resolution imaging tool for mapping the ECM stiffness at length scales sensed by cells. We have developed a novel optical imaging technology, termed laser speckle micro-rheology (LSM) that offers the unique capability to map the micro-mechanical properties of ECM, within the tissue. In LSM, the specimen is illuminated by a coherent laser beam and back-scattered speckle patterns are acquired by a high-speed camera. Spatio-temporal analysis of speckle frames yields the map of viscoelastic modulus, G. Here we demonstrate the capability of LSM for micro-mechanical mapping of the tissue through imaging a micro-structured phantoms and a normal human breast tissue specimens. These results open new avenues for investigating the mechano-biological mediators of disease and development in the future.

Currently there is no accurate objective measure for monitoring pain during the state of drug-induced unconsciousness (such as during surgical anesthesia). Moreover, the absence of an objective measure for detecting pain hampers the physician's ability to provide an optimal dose of analgesics. We have developed a novel method for detecting pain by quantifying skin blood flow dynamics using a miniaturized dynamic light scattering (mDLS) sensor placed on the skin. Healthy awake volunteers were studied with mDLS sensors placed on both index fingers while being subjected to a series of cutaneous painful stimuli (electric shock and heat), randomly applied in a range between the subjects’ pain threshold and tolerance. Power spectrum analysis of the recorded signal was performed with a focus on two frequency bands, representing relative blood flow of non-pulsatile vessels and larger pulsatile arterioles. Relative blood flow of pulsatile vessels decreased while flow of non-pulsatile vessels increased in response to painful stimulation, with a high correlation between the responses obtained on the right and left index fingers. The changes in hemodynamics that occur during painful stimulation suggest a redistribution of blood flow between pulsatile and non-pulsatile vessels, probably related to central activation of the sympathetic system combined with local dynamic autoregulatory responses. Thus, optical parameters of skin blood flow can detect nociceptive stimuli and consequently can serve as objective biomarkers of pain.

Flow cytometry is the main technology used in hematology analyzers. However, this technology requires bulky and complex hardware systems. Lens-free imaging is an emerging microscopy technique based on a simple and compact inline holography setup. This technique enables to image a large field-of-view (≈30mm²) leading to statistical counting (>10 000 cells) in a single-shot acquisition consistent with performances required in hematology. We report high accuracy platelet count in 54 platelet-rich plasma samples. This accuracy can be achieved through a wise choice of the illumination spectral properties and an optimized algorithmic chain dedicated to small pure phase objects.

Self-measurement of blood pressure (BP) is important for monitoring treatment of hypertension, but current instruments are cumbersome and at times also impractical, especially for the older population. Current optical solutions, such as PPG-based technologies that were developed for improving convenience, provide derived measurements that are often inaccurate, particularly for diastolic values. Alternatively, by using dynamic light scattering (DLS) we are able to measure the direct hemodynamic response. We propose a simple physical model that explains the relation between arterial pressure values and the hemodynamic response which is measured from the finger root following changes in externally applied pressure. Based on this model we have developed a small-scaled, optical, mobile device that measures BP at the finger using dynamic light scattering. The apparatus is positioned at the base of the index finger and contains a ring with an inflatable cuff with two miniaturized dynamic light scattering (mDLS) sensors situated distal to the cuff. The cuff is inflated to above systolic pressure, and changes in blood flow (hemodynamics) are measured during cuff deflation. BP measurement is carried out using specially designed algorithms based on hemodynamic indexes and waveform analysis which capture systolic and diastolic points in real-time. Using this apparatus, we measured BP from 69 patients visiting a hypertension outpatient clinic, and a control group of 15 healthy subjects. BP readings were compared with measurements recorded at the arm location with an Omron device used in the clinic. The mean absolute error (MAE) for systolic and diastolic blood pressure was 7.8 and 9 mmHg, respectively at all ranges of BP measured. In conclusion, using Elfi-Tech's innovative technology, it is possible to measure BP accurately at the finger location using a compact, convenient mDLS-based device with high accuracy.

Surveying disease vectors is currently excessively laborious for continuous and widespread monitoring. Wing beat modulation spectroscopy gives opportunity for species and sex recognition in electronic traps or mosquito target classification in lidar. We used a polarimetric dual-wavelength-band laboratory system to record kHz modulated backscattered light from insects. The system operates in the near and short-wave infrared at 808 nm and 1550 nm and retrieves both co- and depolarized light. Here we give clues on the harmonic content and covariance of four mosquito species and fruit flies. Further, we interpret the interdependence of harmonic strengths when insects transit the probe volume with random heading direction and provide correlation matrices for coherent and incoherent light. Using the obtained parameters, we demonstrate that species that are difficult to distinguish with microscope can be classified with high accuracy. The results are valuable for understanding wingbeat harmonics in relation to heading and valuable for optimal sensor design for disease vector surveillance.

The interest to the use of polarized light in various modern biomedical applications is significantly growing. We explore the influence of scattering and birefringence on the phase shift between electric field components of polarized light propagated through biotissues. Degree of polarization and phase shift between the orthogonal components of circularly polarized light, propagated through the tissue samples, are examined utilizing Poincaré sphere. Scattering reduction and birefringence increase are achieved, respectively, by optical clearing and mechanical stretch. Notably different signatures of state of polarization are observed for scattering and birefringence alterations that makes it possible to distinguish mechanisms of phase retardation.

The design, fabrication and characterization of phosphate based bioresorbable optical fibers is reported. Applications in diffuse optics, pH sensing and temperature sensing have been demonstrated paving the way to the use for a new generation of implantable and degradable devices for theranostics.

The process of tumor growth is a phenomenon which, if understood better, could greatly improve the diagnostic and treatment of patients. In this context, the use of polarimetric imaging can offer more information than classical imaging methods. Here we use a new kind of calibration-free spectro-polarimeter which can be helpful for optical biopsy. Fifty different mice have been studied in full Mueller polarimetry with this device. Some were injected with very pigmented melanoma cells, others with non-pigmented breast cancer cells. Variations in depolarization were measured throughout this study: melanomas were accompanied with intense drops in depolarization, while mice injected with breast cancer cells showed a more diffuse decrease in depolarization. The study confirmed the potential of polarimetric imaging as an optical biopsy tool.

Due to the high flexibility and throughput, Spatial Light Modulator (SLMs) have been widely used in optical microscopy and spectroscopy for various applications. In this talk, I will present some example applications of Raman spectroscopy using two types of SLM, LC-LSM (liquid crystal-spatial light modulator) and DMD (digital micromirror device). Several modalities of Raman spectroscopy can be achieved by using LC-LSM and/or DMD, such as multi-foci Raman spectroscopy and spatially offset Raman spectroscopy (SORS). The SLMs allow the system to be flexible and efficient in collecting Raman signal, requiring no changes to the optical system or mechanical adjustment.

Optical detection techniques based on surface enhanced Raman spectroscopy (SERS) can provide relevant information on molecular and protein composition of biological samples, thus enabling to discriminate between physiological and pathological conditions. With the attempt to develop point-of-impact diagnostics devices, in this study we combine lowcost fabrication processes with surface functionalization strategies for the fabrication of SERS-active polymeric substrates engineered to selectively detect specific biomarkers. By reversibly coupling these devices with the distal end of portable Raman instruments, SERS measurements could be potentially implemented for the early diagnosis of widespread pathologies by SERS analysis of liquid biopsies.

Cardiac surgical patients with cardiopulmonary bypass receive large heparin doses to prevent thrombosis during surgery. Activated clotting time (ACT), used to assess anticoagulation, correlates poorly with heparin plasma concentration and lacks information on key coagulation metrics such as fibrin polymerization (α-angle) and clot strength (MA). Here we assess the accuracy and measurement sensibility of our novel optical sensor, iCoagLab, in measuring several coagulation parameters including ACT, α-angle and MA and evaluate its capability to monitor anticoagulation during cardiac surgery. iCoagLab measures anticoagulation by assessing changes in blood viscosity from intensity fluctuations of laser speckle patterns measured from 25μL of blood sample. In this study, blood samples from 9 volunteers spiked with increased concentrations of heparin (1-5USP/mL) and from 30 patients undergoing cardiac surgery were assessed using iCoagLab. Coagulation parameters, including, ACT, α-angle and MA, were extracted and compared with corresponding results obtained from thromboelastography (TEG), ISTAT-kaolin-ACT and Hepcon-HMS-Plus-instruments. In volunteer samples, heparin treatment significantly prolonged the ACT values measured by iCoagLab which correlated closely with TEG (r=0.91, p<0.0001) and ISTAT-ACT (r=0.78, p<0.0001). At high heparin dose, both the iCoagLab and TEG presented a decrease in MA (p<0.01). Similarly, in cardiac surgical samples, iCoagLab-ACT highly correlated with Hepcon (r=0.76 p<0.0001) and TEG-ACT (r=0.86, p<0.0001). Furthermore, the iCoagLab and TEG MA and α-angle were also significantly modulated by surgery (p<0.05-0.0001). In conclusion, iCoagLab accurately measured anticoagulation and global hemostasis using a drop of blood, likely opening the unique opportunity for multifunctional coagulation monitoring at the point-of care during cardiac surgery.

To evaluate, calibrate equipment, and check the safety of THz devices etc, biotissue phantom is needed for these purposes. Although various researches about biotissue phantom using water have been done, such phantoms are not ideal. Because of the evaporation of water, the optical properties of a phantom change as the time goes by, since THz radiation is very sensitive to the water concentration of the sample. We chose graphite as the substitute of water, and therefore the water-free biotissue phantom was developed to mimic the similar optical properties as human tissues. In order to determine the concentration of each component precisely, quantitative analysis is needed. In this work, we used several mathematical models of the effective medium theory, including the Polder and van Santen model, the Landau, Lifshitz, Looyenga model, the model of complex refractive index, and the Bruggeman model, to study the influence of different graphite concentrations on the refractive index of the water-free biotissue phantom. Phantoms with different graphite concentrations were simulated and 3 phantoms with different graphite concentrations were produced to evaluate the reliability of each model. The fabricated phantoms were then compared with stomach tissues. The result also shows the promise that by using the proper mathematical model, correct concentration can be calculated for other tissue phantom.

The illusive nature of optical trapping dynamics under high repetition-rate femtosecond pulsed excitation has recently been theoretically explained based on nonlinear nature of force and potential arising from the optical Kerr effect. Here we present experimental results of trapping of 1 μm polystyrene beads probed by analyzing back-scattered signal from realtime video microscopy which is helpful in studying the time control dynamics of micron-sized objects suitable for biological applications.

Foodborne disease is one of the major public health problems worldwide. The conventional methods used to detect foodborne pathogens require multiple incubation steps and take a long time to results. Thus, there is an urgent need for biosensors that can determine pathogens without special sample preparation. This paper outlines a method for discriminating E. coli contaminated meat since this bacterium has been implicated as one of the main causative agents of food illness. The method is based on the laser-induced fluorescence response of the Flavin group that is presented among E. coli bacteria’s metabolites. The fluorescence activity of metabolites produced by five E. coli strains was investigated. The list of analyzed strains contains ATCC 25922 E. coli, Enteropathogenic E. coli, Enteroinvasive E. coli, Enterotoxigenic E. coli, and Enteroaggregative E. coli. The preliminary results have allowed developing the statistical model using a part of the fluorescence spectra in the range of 520-560 nm when excited at 450 nm. The proposed model is aimed to differentiate contaminated and uncontaminated meat samples. It has been confirmed that the examined technique provides detection of a bacterial concentration of 10⁶ CFU∙cm-² in five hours after initial contamination at room temperature. The further improvement of the method by using fluorescent probes is discussed.

Upon polarimetry the polarization state of light can be obtained for different depolarizing or non-depolarizing medias such as biological specimens. Tissue polarimetry can facilitate a differentiation between healthy and (pre)cancerous tissues, without using any contrast agents and ionizing radiation. Early cancer detection is vital to increase the life expectancy of patients. Turbid medias like biological tissues can change the state and/or decrease the initial polarization of light. Circularly polarized light is preferred as found to possess better ability to detect abnormal changes in tissues, compared to linearly polarized light. In this paper we analyse polarimetric parameters, measured with Thorlabs Stokes meter included in tissue polarimetric experimental set- up in reflection geometry and multiple ex vivo colon samples. The polarimetric device operates in the spectral range between 400 nm and 700 nm, where all experiments had been conducted with wavelength of 635 nm. By reaching reference values for the polarimetric parameters we can propose a theoretical Mueller matrix, that can be used to describe the depolarization properties of the colon samples used in the experiments. The proposed Mueller matrix is to be modelled and experimentally validated to find out if it matches the theory and can be further decomposed to three matrices of depolarization, diattenuation and retardance. All of the aforementioned experimental approaches are a step closer to a pre-clinical trial, which is a bridge to the final and the most challenging goal - tissue polarimetric set-up for in vivo diagnostics.

Multichannel Fiber Optic Probes (MFOP) is new type of accessories’ which can be used for Modern Biophotonics Techniques, based on: UV-VIS- NIR spectroscopy, fluorescent spectroscopy. New simulations program MFOBv.3 for MFOP allow to specify basic reflex light optic value I0 as a function of distance Z[μm] have been developed. MFOBv.3 make possible quick selection and optimization of the probe fiber bundle structure and fibers specification. Designed Reflex probe for endoscopy spectral evaluations include new type of fibers. Fibers with core surrounded by hollow circular guide provide necessary wide numerical aperture. Multimode metal coated fibers used in MFOP for tumor diagnostic in surgery. Symbioses of Microtechnology and new types of fiberoptic allow made smallest Reflex probe 7+1 fibers structure (OD=0.6mm). Designed sensitive electronic system with 405nm/50mW laser synchronized with fluorescent signals measurement device. For tumor diagnostic during stomach surgery created high sensitive endoscopic fiber multichannel probe for special photosensitizer with excitation wavelength 405 nm. Reflex probe provide sensitivity small than 10 microgram photosensitizer for 1 kg biomass. Experimental results for bio liquids and for tissue with laser induced fluorescence diagnostic extended in poster/report.

In the last decade, multimodal imaging raised increasing interest to overcome the limits of single techniques and improve the diagnostic potential during the same examination. This gives rise to the need for phantoms and procedures for standardizing performance assessment of the multimodal instrument. The SOLUS¹ project adopts this methodology with the aim to build a multimodal instrument (based on diffuse optics -DO-, shear wave elastography -SWE-, and ultrasound imaging -US-) to increase the specificity of breast cancer diagnosis. Here we propose a long-lasting phantom based on silicone material (easier to manipulate with respect to other material for bimodal phantom such as polyvinyl alcohol, PVA) and suitable for both diffuse optical imaging/tomography and ultrasound acquisitions, designed within the SOLUS project. To achieve this goal, we explored a new silicone material for diffuse optics and ultrasound (Ecoflex 00-30), creating a new fabrication recipe and demonstrating its suitability for multimodal imaging if coupled to another silicone elastomer (Sylgard 184), featuring similar optical and acoustical performances except for the echogenicity. The main advantage of the proposed phantom is the capability of tuning independently optical and acoustical performances, thus allowing one to mimic a wide range of clinical scenarios.

We applied reflection-mode terahertz (THz) pulsed spectroscopy to study ex vivo the optical properties of human brain tumors with the different World Health Organization grades, as well as of perifocal regions comprised of intact (healthy) and edematous tissues. We applied gelatin-embedding in order to fix freshly-excised tissues, thus, preserving them from hydration/dehydration and sustaining their THz response unaltered for a couple of hours after resection. We observed a contrast between the THz optical properties of intact tissues and tumors, including gliomas and meningiomas of the brain, in turn, the response of edematous tissues is close to that of a tumor. The observed contrast between intact tissues and tumors has an endogenous character and originates reportedly from increased water content in a tumor due to edema, abnormal vascularity and, in some cases, necrotic debris. The observed results justify a prospect of THz technology in the intraoperative label-free diagnosis of human brain tumors.

In this study, confocal Raman spectroscopy revealed biochemical differences between single normal red blood cells and poikilocytes. The intensities of 5 Raman bands associated with oxygenated RBCs are larger in acanthocytes than normal and normal-looking RBCs. Overall, acanthocytes carry more haemoglobin and this is corroborated by CBC and ICP-MS.

We propose functionalized graphene oxides as an effective cancer cell-targeting agent for multiphoton imaging and nearinfrared photothermal therapy. The dual-functional property of functionalized graphene oxides is investigated in human breast cancer cell lines (MDA-MB-231).

Laser Speckle Contrast Imaging (LSCI) is a powerful low-cost method for visualization of flow, microcirculation and blood perfusion. Due to the fact that diseased and healthy tissues has different blood perfusion, LSCI can be a perspective tool for cancer diagnostics and discrimination between different types of tissues. Previously, multispectral diffuse reflectance imaging method for melanoma diagnostics has been introduced. In this work, multi-wavelength (532-, 655- and 850- nm) LSCI technique combined with hyperspectral camera and diffuse reflectance imaging method will be used for assessment of tissues with different skin perfusion properties. An in vivo experiment with occlusion in human finger was performed serving as a model of tissues with different perfusion properties. The proposed method still requires further development and improvements to become a real clinical laboratory tool for non-invasive skin cancer diagnostics.

Adsorption of photodynamic active dyes (hypericin, chlorin e₆, methylene blue, ALA and fluorescein) and they release from clinoptilolite type of zeolite (CZ) in the presence of biomolecules, such as collagen, albumin and hemoglobin, were quantitatively investigated by absorption and fluorescence spectrometry. Using multiphoton microscopy, we demonstrated that effective inducing of two-photon excited luminescence and second harmonic generation signals in nano/micro-particles of CZ by femtosecond near-infrared laser excitation can be successfully utilized in multiphoton imaging of CZ particles and the dye adsorption processes. In addition, CZ magnetic (MCZ) particles were fabricated and proposed as a promising material for drug delivery and controlled release in biological systems. It is shown that the temperature of the aqueous suspension of MCZ of 2 cm³ can be increased by 7 ºC for 3 minutes by an induction heater. Furthermore, it was demonstrated that MCZ particles have strong inhibition and destruction effects on insulin and lysozyme fibrillization, and the protein amyloid formation is inhibited in a dose-dependent manner. Optical spectrometry and multiphoton microscopy are effective approaches that may reveal potential of magnetic zeolites in drug delivery and biomedical imaging of cancer photodynamic therapy and hyperthermia.

The main purpose of the presented studies were both cytotoxicity studies of the developed hollow gold nanoshells and examination of their applications as photothermal agents. We decided to used hollow gold nanoshells as photoactive agents in photothermal therapy (PTT) due to their suitable optical properties, which are essential for this kind of tumor therapy. Nanoparticles were obtained by the galvanic replacement method. We confirmed excellent optical and photothermal properties of surface modified (with PEG-COOH and PEG-NH₂) nanoparticles. Then cytotoxicity studies were conducted on two selected human cell lines: A375 (malignant melanoma) and HaCaT (aneuploid keratinocyte). Cells viability was evaluated using MTT test. Finally, we evaluated effectiveness of PTT based on AuNPs-PEG-COOH or AuNPs-PEG-NH₂.

We have designed a fiber probe based optical diagnose system for detection of interspecies transmissibility. We have showed the optical performance to measure the optical signal of the target sample by using the manufactured fiber probe. We have confirmed the capability of our system to be utilized to biomedical diagnose applications.

Bone healing involves compositional changes and understanding these is potentially important for prevention and treatment of bone diseases like osteoporosis. The health and proper function of bone tissues depends crucially major elements like calcium and phosphorus, but also on trace elements like iron, zinc, and strontium. This work employs energy dispersive X-ray fluorescence spectroscopy (μ-EDXRF) for trace element analysis, particularly of iron, and Raman spectroscopy for analysis of collagen, during healing of subcritical calvarial defects. In vivo defects were created on the calvaria of Sprague-Dawley rats (n=16) using a 1.4 mm burr drill. Subjects were sacrificed and additional control defects were similarly created after 7 (n=8) and 14 days (n=8) of healing. The two spectroscopy methods analyzed the bone surface at both time points and defect types without mechanical perturbation. Compared to control defects, calcium was found significantly decreased while zinc and iron increased in in vivo defects. Iron increased 6.7fold after 7 days, and this increased reduced approximately 50% after 14 days. Raman showed decreased collagen alignment after 7 days, which became insignificant after 14 days. We deduce that new collagen formation during healing, as revealed by Raman spectroscopy, scanning electron and optical microscopy and surface profiling, resulted in trace element changes. Our results show the need for studying the concentrations of major and trace elements, with iron in particular playing a crucial role in healing.

We have recently introduced a novel methodology for noninvasive assessment of structure and composition of human skin in vivo. The approach combines pulsed photothermal radiometry (PPTR), involving time-resolved measurements of midinfrared emission after irradiation with a millisecond light pulse, and diffuse reflectance spectroscopy (DRS) in visible part of the spectrum (400–600 nm). The experimental data are fitted simultaneously with respective predictions from a four-layer Monte Carlo (MC) model of light transport in human skin. The described approach allows assessment of the contents of specific chromophores (melanin, oxy-, and deoxyhemoglobin), as well as scattering properties and thicknesses of the epidermis and dermis. However, the involved multidimensional optimization with a numerical forward model (i.e., inverse MC, IMC) is computationally very expensive. In addition, each optimization task is repeated several times to control the inevitable numerical noise and facilitate escape from local minima. Thus, assessment of 14 free parameters from each radiometric transient and DRS spectrum takes several hours despite massive parallelization using CUDA technology and a high-performance graphics card. To alleviate this limitation, we have developed a computationally very efficient predictive model (PM) based on machine learning technology. The PM is an ensemble of decision trees (random forest), trained using ~10,000 "pairs" of various skin parameter combinations and the corresponding PPTR signals and DRS spectra, computed using our forward MC model. While the parameter values predicted by the PM are very similar to the IMC results there are some concerns regarding their accuracy. Therefore, we present here a hybrid model, which combines the described PM and IMC approaches.

We studied the influence of short-term heating/stretch-fixing stimulus on smooth muscle cells in vitro to determine the condition of optimum vascular dilatation for our novel angioplasty. A short-term thermal angioplasty named Photo-Thermo Dynamic Balloon (PTDB) has been proposed for stenotic artery treatment. Stretch-fixed smooth muscle cells after PTDB dilatation was reported previously. This formation would be a risk of restenosis because of enhancement in smooth muscle cells’ migration ability. To estimate migration ability after short-term heating/stretch-fixing, we studied respiratory activity of smooth muscle cells up to 96 hours in vitro. Smooth muscle cells were heated (50ºC or 60ºC, 15 s) with stretching (0 or 40%), and fixed. We measured respiratory activity of smooth muscle cells and alive cell number at 48 and 96 hours after short-term heating/stretch-fixing. A WST-8 assay solution and nuclear stainings (Hoechst 33342 and Propidium Iodide) were used for measurement of the respiratory activity and alive cell number, respectively. The absorbance value at 450 nm after WST-8 assay divided by the alive cell number was used for activity evaluation. We obtained that the respiratory activity of smooth muscle cells after short-term heating/stretch-fixing was drastically decreased in the case of 60ºC heating. In the case of 50ºC heating, the respiratory activity of smooth muscle cells was decreased more gradually than that of 60ºC. We think that heating smooth muscle cells at 60ºC in PTDB dilatation might sufficiently suppress the cell migration. Chronic restenosis suppression might be suggested in that case.

A novel LED based light source with a high degree of modularity is resented as a means of illumination in a hyperspectral imaging system, employing multiple LED panes with different wavelengths. We report on first results and experience gained using such illumination in a large area hyperspectral imaging system.

Colorectal cancer is the fourth-most prominent cause of cancer related fatalities across the globe. Conventional electrocautery techniques used in the resection of colon tissue cause a relatively high degree of collateral damage to the healthy tissues bordering the target sites. Ultrafast infrared lasers offer significantly improved localisation in the ablation of such biological tissues arising from a plasma-mediated ablation mechanism. This improved localisation is two-fold, with lateral confinement and precise depth control being advantageous in minimising thermal necrosis and avoiding bowel perforation respectively. Various laser scanning strategies and optical elements have been investigated, with the intent to exploit the inherent advantages offered from applying photonics to these procedures. Evaluation of the corresponding ablation characteristics was carried out using three-dimensional optical profilometry and histological analysis. If adopted in operating theatres, surgeons could benefit from more control when carrying out resection of neoplasia in the mucosal or submucosal layers of colon tissue, compared to previous electrocautery methods.

Study of bruise characteristics and evolution is of much interest in forensic sciences, with many objective techniques being researched. In this study we combine the optical methods of diffuse reflectance spectroscopy (DRS) and pulsed photothermal radiometry (PPTR) to measure signals from healthy and bruised skin. From these measurements we first obtain initial physiological parameters for a four-layer model of healthy skin near the bruised site. A bruise model is constructed by inserting a blood pool into this baseline model to simulate a bruise followed by bruise dynamics simulation for PPTR signals of bruises. Obtained bruise dynamics parameters describe the evolution of the bruise. The results show that the choice of a suitable healthy baseline affects bruise parameters obtained by fitting the simulated signals to the measurements. By using healthy skin baselines with similar melanin and papillary blood fractions during analysis, comparable bruise parameters are obtained. Differences in layer thickness and scattering properties of healthy skin did not significantly influence these parameters. In contrast, higher papillary blood content in one site resulted in considerably different bruise parameters. Our findings show the importance of good determination of a healthy baseline, preferably using the baseline obtained by a simultaneous fitting of multiple measurements.

Laser speckle patterns were used for characterization of skin topography (DermaSpec, 13N13841). A set of parameters were obtained for skin surface characterization. Preliminary data has already shown differences between skin tumor and healthy skin areas.

The paper proposes an approach of a novel non-contact optical technique for early evaluation of microbial activity. Noncontact evaluation will exploit laser speckle contrast imaging technique in combination with artificial neural network (ANN) based image processing. Microbial activity evaluation process will comprise acquisition of time variable laser speckle patterns in given sample, ANN based image processing and visualization of obtained results. The proposed technology will measure microbial activity (like growth speed) and implement these results for counting live microbes. It is expected, that proposed technology will help to evaluate number of colony forming units (CFU) and return results two to six times earlier in comparison with standard counting methods used for CFU enumeration.

Visible to near-infrared spectroscopy have been applied for non-invasive assessment of meat freshness. The reduction of oxymyoglobin absorbance associated with freshness drop is clearly seen in the visible range of spectra, as well as supplementary fat, water, and proteins contents variations are observed in the near-infrared range. A table-top spectrophotometer equipped with an integrating sphere was utilized for a shallow probing depth (80 μm) and covered 400-1700 nm spectral range. A fiber-optic linear array was coupled to a portable spectrophotometer (measurement range 400-1000 nm) for increasing the average probing depth up to 570 μm. The studied samples of meat experienced an immediate loss of superficial freshness, while kinetics of spoilage was detected after about 2.5 hours. The fiber-optic approach capable for sensing freshness and spoilage process shows promise for design of a compact, portable device for a variety of users at the meat supply chain.

The detection of molecules by surface-enhanced Raman spectroscopy (SERS) is dependent on the nanomaterial used to induce the enhancement effect. This depends on a variety of parameters of the substrate such as the metal used for their creation, their shape, size and size distribution, concentration, as well as the parameters of the solution, such as packing of the nanoparticles, the complexity of the sample, the solvent, etc. It is most crucial, that the parameters are kept constant to provide uniformity of the enhancement. this is crucial for the development of SERS as a reliable and quantitative technique for bioanalysis. Here, we have developed the silver-core and gold-shell nanoparticles, to serve as the enhancement material. The fabrication phase involved constant concentrations of chemicals stability of the solution physical parameters like stirring and heating, and differed only in the perturbation of the reagents addition kinetics. These nanoparticles were investigated further with their ability to measure the solutions of 2-naphtalenethiol in DMSO, as model for testing the variability of the signal due to the enhancement and the kinetics of the nanoparticle-sample solution during a routine Raman measurement procedure. The results indicate vast difference in the preference of the 2-naphthalenethiol to come into contact with the nanoparticles and the partial enhancement of DMSO in most cases, with an almost complete by-pass of the solvent and direct detection of the 2-naphthalenethiol in one case. Moreover, the kinetics of the measurement solution, or its stability during measurement, is provided.

In this work, we developed a kind of gold-silver alloy film based surface plasmon resonance (AuAg-SPR) biosensors with wavelength interrogation to detect C-reaction protein (CRP) by using gold nanoparticles (AuNPs)-enhanced sandwich immunoassay, and the limit of detection (LOD) of CRP was found to be 5 pg/ml . In conclusion, using the AuNPsenhanced sandwich immunoassay and the SPR chip of AuAg alloy film can detect CRP effectively and reduce the LOD significantly, and this improvement can also be applied for the detection of any biomarkers in low concentration accurately.

A new method of acousto-optic processing of electrocardiographic signals, especially obtained in the ultra high resolution mode, has been proposed, and its information possibilities have been considered. It has been shown that elecrocardiographic signals processing can be performed only by acousto-optic spectrum analyzers with time integration or with time-and-space integration. In order to provide the necessary resolving power by frequency which corresponds to the frequency range of bioelectric signals to be studied, it is necessary to make some specific demands to the accumulation time and to amount of pixels of the multielement photodetectors used in acousto-optic devices mentioned above. The proposed method provides the significant shortening of the processing duration and improvement of the processing reliability. The distortions which appear due to the finite size of the photodetecting array pixels have been discussed and some means have been proposed in order to minimize the distortions and to increase output signal-to-noise ratio. The method can provide early diagnostics of ischemic heart disease.

Among semiconductor quantum dots, silicon nanocrystals (SiNCs) are gaining interest due to their high biocompatibility and natural abundance of silicon. The optical properties make SiNCs optimal candidates for luminescent bioprobes: (i) emission energy tuneable to the red and NIR spectral region, compatible with the biological window; (ii) high photoluminescence quantum yield; (iii) no sensitivity to molecular oxygen despite the long lifetime of emission; (iv) long-lived luminescence that enables time-gated detection in the hundreds of μs timescale, which allows removal of scattered excitation light and autofluorescence of the biological sample with a low-cost equipment (gating times of the order of hundreds of μs). SiNCs can be passivated through covalent bond formation between Si and C atoms giving extremely robust systems. The two major issues for SiNCs are the poor absorption outside the UV spectral region and the difficulty to obtain water suspendable SiNCs that maintain their photo physical properties. Our group addressed the first problem by introducing light absorbing units on the SiNCs surface; these dyes can be excited and transfer the energy to the silicon core behaving like a light-harvesting antenna. Regarding the second issue, several attempts are reported in literature to make SiNCs water dispersible, but these approaches often suffer of scarce stability in aqueous environment, and loss of SiNCs photophysical properties. Our approach is based on covalent functionalization of SiNCs via two-step synthesis involving a first coating step in organic solvent and a post-functionalization through thiol-ene click chemistry in order to introduce poly(ethyleneglycol) (PEG) as a water soluble group. SiNCs obtained with this approach are colloidally stable and retain NIR emission with long emission lifetimes (tens of microsecond).