This PDF File contains the front matter associated with SPIE Proceedings Volume 11359, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists

Glioma Stem-like Cells (GSC) are one of the integral parts of Glioblastoma (GBM) and are generally resistant to standard cancer therapy. Radiation therapy is the standard therapy for GBM which improves the survival of patients and GSCs pose a major obstacle in radiation-induced cell death. Though transcriptomic landscape of different GSCs and their radiation response were well studied, the differences in bio-molecular composition are yet to be explored. Raman microspectroscopy being a non-invasive, label free technique, aids to determine the biomolecular constituent of cells at live conditions. Using GSCs with varying radiation sensitivity (training set), we identified inherent Raman spectral signatures specific to different levels of radiation sensitivity. Moreover, tracking irradiation induced changes in GSCs also identified signatures specific to radiation sensitivity. Both inherent and radiation induced signatures predicted the radiation sensitivity with very high accuracy in the test set. Integrated analysis of inherent and radiation-induced alterations through Raman spectroscopy identified differential regulation of several molecules, in particular glycogen, choline and cholesterol between GSCs with different radio sensitivity. Finally, we reversed the resistant phenotype by using small molecule inhibitors specific for the metabolic pathways of these biomolecules. Thus, we have found Raman spectral signatures to predict radiation sensitivity of GSCs. Further, we also demonstrate radiation-induced molecular changes in the GSCs and methods to reverse radiation sensitivity by using small molecule inhibitors. These findings will have clinical application in radiosensitizing the GSCs. Efficient anti-tumor therapy can be attained with lower dosage of radiation along with these inhibitors with less side-effects.

Rapid, non-invasive methods for the detection and identification of pathogens are becoming very important in the field of medicine and defence. There is a constant need for technologies that can rapidly detect and identify the presence of airborne, foodborne or waterborne pathogens and toxins with high precision. Conventional methods employed by microbiologists include phenotypic and genotypic methods. The most commonly used biochemical methods include enzymatic activity, gas production and compound metabolism for identification of bacteria. Most of the methods mentioned above are time consuming and require extensive sample preparation. Raman spectroscopy is a well established molecular spectroscopic technique that measures bond vibrations to decode molecular structure and chemical composition of samples. In the realm of biochemical analysis it is essential that a technique is non-invasive, preferably label-free and non-destructive. These unique features make Raman spectroscopy and its variants an important tool for identification of samples. However, biological samples are very complex as even a single cell or a bacterium is composed of a number of biomolecules such as proteins, lipids, carbohydrates and nucleic acids. Raman spectroscopic analysis yield spectral fingerprint unique to the biomolecules. Therefore, these spectral markers can be used for tracking diseases, studying effectiveness of drugs in cells and tissues, identification of pathogens and many other biological processes. Furthermore, accuracy of classification and prediction can be enhanced and automated by combining this technique with chemometric analysis. Sensitivity of detection can be improved employing SERS based approach. Raman spectroscopy based methods have gained popularity in the last few decades due to rapid development in instrumentation that has led to enhanced sensitivity and resolution. However, it has been a challenging issue so far to differentiate and detect pathogenic strains from non-pathogens of the same species. In this manuscript, the work initiated towards identification of pathogens has been discussed. Eight strains of food pathogens were used as a model system and was classified based on PC-LDA using the Raman signals. The classification accuracy was 100%. The potential and limits of Raman spectroscopy based technology for detection of pathogens in real environment has been discussed.

In biomedicine, the mechanical properties of cells and tissues are increasingly recognized as indicator of pathological processes. Established measurement methods do not meet the requirements of biomedicine simultaneously: high spatial resolution, high throughput, non-contact, no markers and low measurement uncertainty. One particularly emerging technique, spontaneous Brillouin microscopy, enables optical three dimensional measurements with high spatial resolution. However, it suffers from low signal to noise ratio (SNR), which results in a long measurements duration. Impulsive stimulated Brillouin scattering (ISBS) is promising to overcome this shortcoming. In ISBS microscopy, an excitation takes place in the measurement volume through the superposition of two beams of an ultra-short pulsed laser. The resulting fringe pattern creates a standing acoustic wave in the sample. This standing wave can be probed with a continuous wave (cw) laser. The deflected part of the probe beam is amplitude modulated and can be measured by a photodiode. The determined center frequency is proportional to the sound velocity in the measurement volume and thus provides information on the local mechanical properties. For experiments, an ISBS microscope was built and characterized. Using this setup, measurements were carried out on the biological model material hydrogel. For the first time hydrogels of different stiffness could be discriminated. According to the requirements of biomedicine, the measurement volume has been reduced in size and thus a lateral resolution at cellular level was achieved. Based on an improved setup, investigations on the minimum measurement duration while satisfying the biomedical requirements mentioned above could be carried. The next steps are the transfer of the measurement technology to an application in biomedicine. A combination with other measurement techniques to a multimodal system might also be useful. In summary, Results obtained in our Research underline that ISBS microscopy offers an enormous potential for biomedical applications.

The aim of this research is maximizing differentiation quality of skin neoplasms by Raman Spectroscopy (RS) and Autofluorescence (AF) using conventional Machine Learning (ML) algorithms (which means excluding Neural Networks). Thus, a basic task of this research consists of making and testing ML algorithm ensembles on Raman spectra, that were obtained in vivo in Samara Oncology Clinical Center. The data (spectra) have been obtained in a form of text files containing the identifier of a patient, as well as a Raman spectrum in the form of pair values – a wavelength and an appropriate value. All data (964 spectra) has been divided for two classes: Tumor and Skin. Further preprocessing of the input data and the analysis of models of machine learning for a problem of classification have been carried out. We used the following ML tools, namely Python 3.7.3, an open source ML libraries Scikit-learn v0.21.2, NumPy 1.17.0, and Pandas 1.0.1, IDE Anaconda Enterprise 5.3 and cloud service Google Colaboratory with an interactive environment Jupyter Notebook. Machine learning models that show a high accuracy result include Classification and Regression Tree (CART), Support Vector Classification (SVC), Logistic Regression (LR), K-nearest neighbors algorithm (KNN). These classifiers show high quality classification on standard parameters already. The Soft Voting Classification module was selected as an ensemble, that allows us to use several models of classifiers that are not similar to each other at once, combine them into one classifier. Results of this ensemble testing from these ML algorithms showed that the classification accuracy, unlike the best qualifier, has not been increased. However, the metrics of classification quality show that the model has become stabler and steady. Results: specificity - 93%, sensitivity - 88, harmonic mean between precision and recall (F1 score) - 90%. An analysis of validation and training curves indicated a small size of training data and, for some cases, a high complexity of the model, which led to a decrease in the classification accuracy.

Advanced wavefront-shaping methods can be used to transform a simple multimode fiber into an ultra-thin laser scanning microscope. Here, we extend this technique to label-free non-linear microscopy with chemical contrast using coherent anti-Stokes Raman scattering (CARS) through a multimode fiber endoscope, which opens up new avenues for instant and in-situ diagnosis of potentially malignant tissue. We use a commercial, 125 μm diameter, 0.29 NA, GRIN fiber as the endoscopic probe. Wavefront shaping on a spatial light modulator is used to create a focus, where the 1-2 ps long pump and Stokes pulses are overlapped in time, which is scanned behind the fiber facet across the sample. The chemical selectivity is demonstrated by imaging 2 μm polystyrene and 2.5 μm PMMA beads with per pixel integration time as low as 1 ms for epi-detection. Epi-detection through the fiber is possible despite the fact that the CARS signal is emitted mainly in the forward direction, away from the fiber facet. Detecting the back-scattered signal from the underlying tissue, requires a large detector aperture to be efficient. By detecting through both the core and the cladding of the fiber, we obtain sufficient detection efficiency.

Tissue morpho-mechanics is gaining an increasing relevance in various fields, including biology, medicine, pathology, tissue engineering, and regenerative medicine, since it targets the relationship between morphological features and mechanical properties in biological tissues, which plays an important role in various biological processes including metastasis, wound healing and tissue regeneration. In particular, in every biological tissue, morphological, biochemical and mechanical properties are tightly connected and they influence each other in a correlative manner. For this reason, a correlative approach employing multiple techniques is ideal for targeting tissue morpho-mechanics with an optical approach. Here we report a correlative study performed by optical microscopies, disclosing the supramolecular collagen morphology correlated with its biomechanical and biochemical analyses. In particular, using human corneal tissue as a benchmark, we correlate Second-Harmonic Generation maps with mechanical and biochemical imaging obtained by Brillouin and Raman micro-spectroscopy, demonstrating that the peculiar mechanical functionality of so-called sutural lamellae originates from their distinctive supramolecular organization. A theoretical model based on the ultrastructural symmetry of corneal lamellar domains provides the interpretation of the experimental data at the molecular scale. The proposed methodology opens the way to the non-invasive assessment of tissue morpho-mechanics and holds the potential to be applicable to a broad range of biological and synthetic materials.

As a non-invasive imaging tool, second harmonic generation (SHG) imaging has shown a great prospect in the visualization of living cells and tissues. It is widely used in the fields of science, medicine, biology and tissue engineering. Although the SHG intensity is often used as the basis of imaging analysis, its interpretation is still very subjective. In this project, the nonlinear process behind SHG is described. In order to explore the wavelength-dependence of SHG, the simulation experiments are carried out. A contrast model is used to calculate the forward- and backward- generated SHG signals. The simulation results show the SHG intensities of different collagen models at the wavelength between 760 nm to 1000 nm.

Sensors in mid-infrared spectroscopy based on attenuated total reflection (ATR) sensing with internal reflection elements (IREs) facilitate easier measurements of aqueous solutions or other opaque analytes. Micromachined silicon (Si) elements are an attractive alternative to conventional IREs, as they can be produced cheaply with silicon processing. Techniques for surface modifications are also easily integrated into the wafer process, and surface structures such as micropillars or nanoparticles can thereby be used for signal enhancement. Replacing the classic Fourier transform infrared (FTIR) spectrometers with tuneable quantum cascade lasers (QCLs) also opens up new avenues for sensing. In this study, the performance of basic and signal-enhanced Si IREs has been compared for measurements in a spectroscopy setup with a fibre-coupled tuneable QCL source. These IREs had V-shaped microgrooves etched on the underside for more efficient in-coupling of light, while the signal enhanced IREs also had micropillars on the top surface. The results are also contrasted with measurements done in a standard ATR-FTIR spectrometer, using an Alpha II spectrometer with a single-reflection diamond ATR crystal. Various concentrations of glucose (0-5000 mg/dl) in aqueous solutions were used to characterise the system performance. The quality of the signal enhancement was evaluated with regard to sensitivity and noise level in the acquired spectra. The microstructured Si IREs gave a signal enhancement of up to a factor of 3.8 compared to a basic Si element, with some concomitant increase in noise. The absorbance was higher for both types of Si IREs as compared to the diamond ATR crystal. The effective enhancement and the limit of quantification improved by a factor up to 3.1 in the signal-enhanced IREs compared to the basic Si element.

Microbial identification is a critical process aiming at identifying the species contained in a biological sample, with applications in healthcare, industry or even national security. Traditionally, this process relies either on MALDI-TOF mass spectroscopy, on biochemical tests and on the observation of the morphology of colonies after growth on a Petri dish. Here is presented an innovative method for label-free optical identification of pathogens, based on the multispectral infrared imaging of colonies. This lensless imaging technique enables a high-throughput analysis and wide-field analysis of agar plates. It could yield very high correct identification rates as it relies on an optical fingerprint gathering both spectroscopic and morphologic features. The setup consists of a Quantum Cascade Lasers light source and an imager, a square 2.72 by 2.72 mm uncooled bolometer array. Microorganisms to be analyzed are streaked on a porous growth support compatible with infrared imaging, laid on top of an agar plate for incubation. When imaging is performed, growth support is put in close contact with the imaging sensor and illuminated at different wavelengths. After acquisition, an image descriptor based on spectral and morphological features is extracted for each microbial colony. Supervised classification is finally performed with a Support Vector Machine algorithm and tested with tenfold cross-validation. A first database collecting 1012 multispectral images of colonies belonging to five different species has already been acquired with this system, resulting in a correct identification rate of 92%. For these experiments, multispectral images are acquired at nine different wavelengths, between 5.6 and 8 μm. Considering the optimization possibilities of the image descriptors currently used and the ongoing development of the uncooled bolometers technology, these very first results are promising and could be dramatically improved with further experiments. Thereby, mid-infrared multispectral lensless imaging has the potential to become a fast and precise Petri dish analysis technology.

Infrared spectra of cells and tissues exhibit a variety of scattering phenomena that have been studied in the literature during recent years. The most intriguing scattering phenomena that have been observed are so-called Mie-type scattering phenomena. Mie-type scattering occurs in nearly spherical-shaped scatterers, when the size of the scatterer is of the same order as the wavelength employed. Mie scattering is the scattering of electromagnetic radiation at a spherical scatterer and was solved analytically by Gustav Mie already in 1908. The analytical Mie solutions have been used in model-based pre-processing approaches for retrieving pure absorbance spectra from highly distorted, measured infrared spectra of cells and tissues. While existing iterative algorithms have been shown to be able to retrieve pure absorbance spectra efficiently in many practical situations, the question remains to what extent the analytical Mie solutions describe the extinction efficiency in practical situations, where the shape of cells deviate considerably from perfect spheres. From other fields it is well known that deviations of shapes can change the absorption properties of a scatterer considerably. In the context of chaos in wave systems, it was shown that chaotic scattering may enhance absorption properties of a scatterer considerably. Small deviations from perfect spherical scatterers that involve changes in the size of the refractive index, may easily lead to a transition between regular and chaotic scattering. The aim of the current study is to investigate how deviations from a spherical scatterer leading to chaotic scattering can change the extinction properties of the scatterer. For this purpose, we investigate a scatterer that is shaped like a Bunimovich billiard. A Bunimovich-billiard shaped scatterer has been shown to be chaotic, and it allows to study the gradual transition between a chaotic scatterer (billiard) and a regular scatterer (sphere).

One of the streams in advanced diagnostic technologies is non-invasive spectroscopic investigation or “spectral histopathology” as a novel alternative for rapid cancer diagnostics and label-free cancer specification. Fiber optics enable faster and more convenient way to study different biological tissues than standard techniques which require destructive sample preparation (e.g. histopathology, chemical analysis). Moving on this direction, we developed and applied various fiber optic probes for key spectroscopy methods such as Raman scattering, Mid IR-absorption, Diffuse NIR-reflection, and auto-fluorescence – to compare them and select the best combination for a real-time detection of malignant tissue in pre-clinical and clinical environment. All four spectroscopic methods have been tested for cancer diagnostics on biopsies of normal and cancer tissues (abdominal, oral and brain), ex-vivo samples and bioliquids. Obtained spectral data were evaluated by multivariate discrimination analysis to enable clear separation of malignant and normal tissues. Benefits of combination of several spectroscopic modalities and data fusion is presented for the better sensitivity, specificity and accuracy. The best synergetic effect 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, several fiber optic probes combining several spectroscopic modalities implemented within the same single probe were designed, assembled and evaluated.

We report a new application of compressional optical coherence elastography (OCE) to discriminate morphological constituents of biological tissues by analyzing OCE images obtained either in vivo or for freshly excised samples. The new technique enables quantitative morphological segmentation of OCE images with delineation of several (~4-6) tissue constituents. As the first step, the method uses compressional OCE to reconstruct stiffness maps for a pre-chosen standardized pressure over the entire area of the OCE image. Then specific stiffness ranges (characteristic "stiffness spectra") are initially determined by careful comparison of the OCE-based stiffness maps with the results of segmentation of "gold-standard" histological slices. After such pre-calibration, the stiffness maps can be automatically segmented into regions, for which the Young’s modulus (stiffness) falls in specific ranges corresponding to the morphological constituents to be discriminated. The results of such automated segmentation of OCE-images demonstrate a striking correlation with the results of conventional segmentation of histological slices in terms of percentages of the segmented zones. High sensitivity of the OCE-method to histological alterations was demonstrated in vivo in comparative studies of various types of anti-tumor therapies using murine tumor models. Studies of >100 samples of freshly excised breast cancer samples revealed strong correlation between the tumor-tissue subtype and its morphological composition determined by the developed OCE method. Thus, the developed approach can be used as a basis for express OCE-based biopsy (feasible intraoperatively). Longitudinal in vivo monitoring of morphological alterations in tumors under therapy or during natural development is also possible for locations accessible to OCT.

This paper reports on line-field confocal optical coherence tomography (LC-OCT), a recently invented imaging technology now capable of generating either a horizontal (en face) section image at an adjustable depth, or a vertical section image (B-scan) at an adjustable lateral position. For both operating modes, images are acquired in real-time (10 frames/second), with real-time control of the depth and lateral positions. Using a supercontinuum laser as a broadband light source and a high numerical microscope objective, an isotropic spatial resolution of ∼ 1 μm is achieved. The imaging fields of view are 1.2×0.5 mm² (x×y, horizontal) and 1.2×0.5 mm² (x×z, vertical). LC-OCT has been used in dermatology for skin imaging.

We use novel possibilities opened by compressional Optical Coherence Elastography (OCE) to characterize both natural interstitial gaps and laser-irradiation-produced porosity in collagenous tissues (corne and cartilage). Under increasing moderate compression up to strains ~several percent, the current Young modulus of cornea gradually increases from initial values below 100 kPa to values >MPa that are closer to Young's modulus of cartilages in which collagen fibers are much denser packed. The lower stiffness of cornea can reasonably be attributed to the initially looser packed collagenous fiber layers, so that initial high compressibility of cornea is dominated by interlayer voids (gaps). By analogy with geophysics, we apply a model describing the reduction in the tissue elastic modulus due to the presence of a system of nearly parallel, thin "crack-like" voids/pores between the collagenous fiber layers. Initially they are highly compressible, but with increasing compression are getting closed, so that the material gets stiffer. Characterization of such porous component in water-saturated packing of collagenous layers in the natural state is inaccessible to AFM and conventional microscopy, whereas OCE enables earlier unavailable possibility to non-invasively characterize such pores/gaps (their total volume and distribution over the aspect ratio) by analogy with crack characterization in geophysics. Also we apply OCE to characterize spatially-inhomogenous modification of pore characteristics by moderate IR-laser-irradiation in regimes typical of collagenous-tissue reshaping. The obtained results are important for obtaining better insight in the structural modificatons in collagenous packings in the context of the development of novel methods of laser-assisted non-surgical methods of cornea-refraction correction and biologically non-destructive reshaping of cartilaginous samples for fabrication of implants in otolaryngology and maxillofacial surgery.

Compressional optical coherence elastography (OCE) is the emerging tool to evaluate Young modulus of the tissue with resolution close to optical coherence tomography. Recent versions of compressional OCE based on the strain comparison within the tissue and the calibrated reference silicone layer (sensor) located between the OCT probe and tissue. For standard realizations of compressional OCE the evaluation of the strain within the tissue and the sensor performed only at one point of the stress-strain curve. In the same way the applied stress can be easily estimated in a wide range on the base on the stress-strain relation for calibrated sensor strain that allow to evaluate the stress-strain relation for the underlying tissue not in one point but in the wider range. In this report we demonstrate this approach to nonlinear parameter evaluation for wide range of the tissues. We demonstrate the application of this new tool to breast tissue, vaginal wall, bladder, brain, cartilages, coronary arteries walls in various states. The preliminary results shows that several states of the tissues such as vaginal wall state in case of pelvic organ prolapse, benign breast tumor and coronary arteries plague can be determined on the base of elastic nonlinear parameters.

This work discusses the design and implementation of a compact forward-looking OCT probe with clinical grade encapsulation that can be readily incorporated into endoscopic systems. Its optical performance makes it particularly attractive for outpatient care use in the optical detection of tissue pathologies inside of the bladder. The use of a tubular piezoelectric-based fiber scanner allows for forward-looking 3D imaging with a single fiber, which can be operated in a quasi-resonant regime to provide a large scanning range with relatively low driving voltages, while maintaining a small outer diameter. This configuration and the adjustable scanning rate of the fiber allow for not only high-resolution OCT images, but also the potential implementation of functional extensions like OCT Angiography. Commercially available focusing optics are used at the tip of the probe, which is designed for telecentric operation in contact with the tissue. The coupling parts, as well as the probe housing, are produced using selective laser-induced etching of fused silica, a 3D structuring approach that enables the creation of alignment and pinning features with micrometer precision. The use of this technology allows for finely tuned optical and mechanical designs, while facilitating the mounting and interconnection of components, providing a reliable assembly process.

Lasers have introduced many advantages to the medical field of osteotomy (bone cutting), however, they are not without drawbacks. The thermal side effects of laser osteotomy, in particular, affect a patient’s healing process. Employing an irrigation system during surgery is a standard solution for reducing thermal damage to the surrounding tissue, but, due to the high absorption peak of water at the wavelength of Er:YAG laser (2.96 μm), accumulated water acts as a blocking layer and reduces the ablation efficiency. Therefore, irrigation systems would benefit from a high-speed and accurate feedback system to monitor the temperature changes in the tissue of interest. Phase-sensitive optical coherence tomography (PhS-OCT) is a highly sensitive method for measuring internal displacement (photothermal-induced expansion) during laser surgery. In this study, we utilized the integrated swept source PhS-OCT system (operating at a central wavelength of 1314 nm and with an imaging-speed of 104,000 A-scans/s) with an Er:YAG laser to detect localized phase changes induced by laser ablation irradiation and thereby quantify the photothermal-induced expansion of bone. The PhS-OCT system was calibrated by measuring the phase changes corresponding to the displacement of cover glass attached to a piezoelectric actuator (PA4HEW, Thorlabs) at different operating voltages. Furthermore, we explored how the induced photothermal expansion of bone changes when irradiated by different pulse energies. Using a PhS-OCT system to spatially and temporally resolve measurements of axial displacement of bone during laser surgery can play an important role in determining the corresponding temperature map, which can, in turn, offer feedback to the irrigation system in smart laser osteotomy.

We develop a model of full-field optical coherence tomography (FF-OCT) that includes a description of partial temporal and spatial coherence, together with a mean-field scattering theory going beyond the Born approximation. Based on explicit expressions of the FF-OCT signal, we discuss essential features of FF-OCT imaging, such as the influence of partial coherence on the decay of the signal with depth that is captured by the model. We derive the conditions under which the spatially averaged signal exhibits a pure exponential decay with depth, providing a clear frame for the use of the Beer-Lambert law for quantitative measurements of the extinction length in scattering media.

Mid-infrared optical coherence tomography (MIR OCT) has shown promise in the last few years in applications such as spectroscopy and non-destructive testing. Previously, we have successfully demonstrated a MIR swept-source OCT and measured its noise from three main sources: quantization noise from the ADC, shot noise from the detectors and relative intensity noise (RIN) of the laser. Of these sources, RIN places an upper limit on the SNR of swept-source OCT systems. We attempt to characterize RIN in greater depth and determine whether it can be reduced through normalization. The pulsed laser used (Block Engineering Lasertune) is tunable within the wavelength range of 5.4-12.8μm. The laser output was held at a fixed wavelength, repetition rate and pulse width. Each laser pulse was integrated to find its average power along the pulse duration. A Fourier transform of the result was used to calculate the ratio of the AC power to DC power, giving a value for RIN. By using a beamsplitter and aspheric lenses to carefully focus the beam onto two detectors (Vigo System’s PVMI-4TE), the two pulse trains can be normalized. Through normalization, RIN was reduced from -74dB/Hz to -92dB/Hz. Increasing the repetition rate and pulse width leads to a decrease in RIN, but an upper limit on the laser duty cycle constrains improvements to RIN via this method. As the swept laser has four integrated quantum cascade laser (QCL) chips, we also examine the effect of different emission wavelengths on RIN.

Ocular blood flow measurement may have a number of potential applications that explore the relationship between blood flow in the eye and diseases such as: diabetic retinopathy, ocular artery obstruction, hypertensive retinopathy and Alzheimer's disease. Reliable and quantitative method for retinal blood flow estimation is still to be created. Doppler OCT is one of candidates for such a method, but suffers from a number of limitations. Recently we proposed a solution to one of the most prominent artefacts in Doppler OCT, which is the phase wrapping problem. This allows for precise recovery of velocity profile the Doppler OCT technique remains sensitive to temporal dependence of the result on the blood flow velocity changing with the pulse during the OCT measurement. In this report we explore this problem and show that the synchronization of the OCT measurement with heart beats only partially gives control over the acquired blood flows.

Unprecedented advances in machine learning have led to a variety of algorithms for the remote evaluation of biomedical images, allowing for cost-effective early detection of diseases. In particular, a lot of efforts are focused on the development of reliable image analysis tools for the early diagnosis of eye diseases. Here we present several new methods for ophthalmic image analysis. We propose a machine learning algorithm for ordering images of the anterior chamber (optical coherence tomography, OCT), which extracts features that discriminate between healthy subjects and patients with angle-closure. We also present an algorithm to detect the OCT images that contain artifacts, and we show that removing these images from the data base improves the performance of the ordering algorithm. Finally, we present algorithms for the analysis of retina fundus images, which are able to segment the vessel network in the retina and extract features from the topological tree-like network structure. We show that these features discriminate between healthy subjects and those with glaucoma or diabetic retinopathy.

Commercial multiphoton microscopes currently include standard and bulk titanium-sapphire laser sources (Ti: Sa) delivering a narrow spectral bandwidth of 10 nm at the full-width at half maximum. Such a spectral width precludes the instantaneous imaging of more than four fluorophores simultaneously in optimal conditions of target. Femtosecond ultrawideband laser system (UWLS) appeared since fifteen years with an approach of spectral broadening of Ti: Sa pulses into a photonic crystal fiber associated with a dispersion compensation system. In the current work, we demonstrate the interest of an alternative laser solution, compact, cheap, simple and turn-key based on a UWLS delivering a unique spectral excitation bandwidth named supercontinuum. We have coupled this system to a passive filtering setup. Thanks to this unique spectral bandwidth, eleven fluorophores are imaged with multiphoton processes without any modification on the excitation parameters.

Platelets are the most important participants in both normal hemostasis and pathological thrombotic process. Platelet activation needs to be studied today because management of this process is the key to progress in the treatment of atherosclerotic cardiovascular diseases. Evaluating platelet activation at the single-cell level is a promising approach for investigating platelet functions, as well as studying the action of various receptors. Previously such single-cell studies were conducted by the immobilization of platelets on the surface, which changes the platelet signaling significantly . In this paper, we describe several activation methods to overcome this limitation, in particular, by use of photolabile “caged” analogues of activation agonists. Activation can be initiated by optical pulse with the duration of tens of milliseconds. Therefore, the technique allows one to track the very early stage of activation in freely moving single platelets. In particular, it enables the assessment of the delay between the stimulus and the calcium response in platelets. The proposed method can be used for in-depth studies of platelet physiology.

Genetically encoded photosensitizers are a unique instrument for investigation of cellular mechanisms of photodynamic therapy (PDT). Fluorescent flavoprotein miniSOG (mini Singlet Oxygen Generator) generates singlet oxygen with a high yield and demonstrates strong phototoxic properties in vitro in cancer cells. However, the effective approaches for PDT with miniSOG have not been developed so far. The purpose of the study is to investigate phototoxic effects of miniSOG induced by continuous wave (CW) or pulsed laser irradiation in tumor spheroids. We found that maximum photobleaching of miniSOG without temperature effects was achieved at 120 mW/cm² in CW mode or pulse periodic mode. PDT in pulse periodic mode provided more pronounced increase in the number of dead cells in comparison with CW mode and, moreover, induced apoptosis more efficiently. Therefore, we report for the first time on an effective regimen for PDT with miniSOG in a tumor spheroid model using pulsed periodic laser irradiation.

Minimally invasive optical systems allow to access deep tissue regions living organisms, for instance for microscopy or cell manipulation. Multi core fibers (CFB) are predestined for advancing needle-size microendoscopy, since they offer several 10,000 independent channels with a total outer diameter down to 0.2 mm. The applicability of CFBs as a means for undisturbed light transfer in optical systems is limited however, since random, unknown and time dependent phase distortions occurs between individual cores. This phase distortion is particularly susceptible to bending and needs to be calibrated in-vivo and without optical access to the distal side. Earlier we reported on a solution based on an in-situ phase calibration using virtual guide stars. To correct the phase distortion, we use a LCoS-based phase modulator (SLM) which simultaneously transforms the CFB into a phased array to generate arbitrary light patterns. We use this technique for endoscopic 3D raster scanning fluorescence microscopy. The scanning process is aided by a galvo mirror and a tunable lens to increase the speed to one Mega voxel (200x200x25) per second. Besides the possibility for imaging as an endoscope the technology can serve also applications like optical stretching, high precision cell ablation and single cell addressing in optogenetics. While precise cell rotation can allow tomographic refractive index reconstruction for instance in cancer diagnostics, the combination with CFBs can enable a translation to in-vivo or lab on a chip application. We demonstrate 2-axis cell rotation with a two-sided optical stretcher and rotation of non-Gaussian mode fields.

In this study, we develop an on-chip optoacoustic flow cytometry (OAFC), which combines multi-color high speed optoacoustic microscopy and microfluidics for cell imaging. The device employs a micro-electro-mechanical-system (MEMS) optical scanner to achieve an ultrafast cross-sectional imaging at a frame rate of 1758 Hz, providing a lateral resolution of 2.8 μm in flowing direction and 2.2 μm in transverse direction. The outer size of OAFC is 30×17×24 mm3. By using multispectral strategy, OAFC is able to resolve particle distribution inside cancer cells, distinguish different cancer cells in a mixture, and screen a few cancer cells from red blood cells (RBCs). The results suggest that OAFC has sufficient sensitivity and specificity for future cell-on-chip applications, including volumetric consistently observation, single cell detection, and clinical cancer diagnosis.

When focusing a light beam at high numerical aperture, the resulting electric field profile in the focal plane depends on the transverse polarisation profile, as interference between different parts of the beam needs to be taken into account. It is well known that radial polarised light produces a longitudinal polarisation component and can be focused below the conventional diffraction limit for homogeneously polarised light, and azimuthally polarised light that carries one unit of angular momentum can achieve even tighter focal spots. This is of interest for example for enhancing resolution in scanning microscopy. There are numerous ways to generate such polarisation structures, however, setups can be expensive and usually rely on birefringent components, hence prohibiting broadband operation. We have recently demonstrated a passive, low-cost technique using a simple glass cone (Fresnel cone) to generate beams with structured polarisation. We show here that the polarisation structure generated by Fresnel cones focuses better than radial polarised light at all numerical apertures. Furthermore, we investigate in detail the application of polarised light structures for two-photon microscopy. Specifically we demonstrate a method that allows us to generate the desired polarisation structure at the back aperture of the microscope by pre-compensating any detrimental phase shifts using a combination of waveplates.

Optical tweezers are powerful scientific tools capable of trapping and manipulating microscopic particles with a tightly focussed laser beam. They can be used to measure nm-sized movements as well as pN-forces in various applications . One of these applications is "dynamic force spectroscopy", where increasing forces are applied to e.g. receptor-ligand bonds to facilitate unbinding. Analysis of unbinding forces, or "rupture forces", can provide an overview of the thermodynamics and activation barriers during bond dissociation. Claudins are considered important component of tight junctions, which form a seal between epithelial cells to regulate paracellular transport. Clostridium perfringens enterotoxin (CPE) is a toxin that binds to certain claudins and disrupts the tight junctions. Understanding of the specific interaction between CPE and receptor claudins could lead to several pharmaceutical applications. This work aims to measure the binding strength between c-CPE and claudins in living cells, using an optical tweezers system. The system was characterized and calibrated extensively, which is necessary for force measurements. The trap stiffness in both lateral directions was measured for 1μm silica particles in cell culture medium. Stiffness values up to kx ≈ 89.81 pN/μm and ky ≈ 129.28 pN/μm in cell culture medium were determined. To investigate the interactions between C-CPE and claudins, 1 μm silica particles were coated with C-CPE and trapped with the optical tweezers. MCF-7 cells, which are known to express receptor claudins were positioned towards the particle until contact, then retracted. The technique can detect unbinding events and measure corresponding rupture forces from 1 pN up to 56.7 pN in magnitude. Overall, this work shows that optical tweezers offer a versatile and precise non-contact method to probe the interaction of transmembrane proteins in living cells.

Microfluidic systems facilitate the realization of compact and miniaturized lab-on-a-chip systems which can be used for various applications. The conventional method to fabricate such devices entails the use of complex etching processes in clean rooms and soft lithography methods which require substantial expertise. Since the design of such microfluidic devices is often customized depending on the application of the user, it would be ideal to have a fabrication technique that would allow for fast and reliable production with the possibility to generate high resolution three-dimensional structures using different materials. 3D printing technique has been recently demonstrated as a means to fabricate microfluidic devices. It enables rapid prototyping of robust and complex structures. Nowadays, 3D printers can create small structures down to several tens of microns. 3D-printed devices can also provide lab-on-a-chip systems which are compatible with optical techniques and microscopy. In this work, we demonstrate the integration of optical manipulation in 3D-printed microfluidic systems with particular focus on optimized design and fabrication protocol. 3D printing was performed using a Multijet printer (MJP2500 Plus). A microfluidic chip was designed for the purpose of dual beam optical trapping and optical stretching of mammalian cells. Three inlets with channel dimensions of 500 µm were used to flow buffer, particles or cells into the device. Two single mode fibers were inserted into fiber guide channels with dimensions of 500 µm, separated with a distance of 300 µm, in order to deliver counterpropagating beams into the trapping region. Hydrodynamic focusing was performed showing that laminar flow can be achieved in the device. In order to evaluate the compatibility of 3D-printed microfluidic chips for optical manipulation, the mean square displacement of the optically trapped 10 µm polystyrene particle was measured for different laser powers. In addition, we demonstrate optical stretching4 of microvascular endothelial cells under flow.

On cellular or tissue level, electrophysiology studies during light stimulation with shaped light can potentially enable the design of effective stimulation strategies towards future cardiac therapies. In this work, we explore the use of optogenetics in probing the electrophysiology of a functional cardiac syncytium. A holographic technique with the use of a spatial light modulator allows beam shaping and precise spatiotemporal control of the origin of action potential in a cardiac-like cell line monolayer. Depolarization was performed by using a blue laser (488 nm) incident to a reflective ferroelectric liquid crystal on silicon spatial light modulator (FLCOS-SLM) which generates multiple foci on to cardiomyocyte-like cells (HL1 line) expressing the light-gated protein Channelrhodopsin-2 (ChR2). The calcium-sensitive fluorophore Cal-630 was used to visualize the cellular electrical activity and thus can be combined with ChR2 as an all-optical method to stimulate and visualize electrophysiology of cardiac-like cells in a non-contact manner. We generated single or multiple foci each with spot size of ~5 µm for optogenetic stimulation. The probability of triggering an action potential, which we refer to as excitation probability, is highly dependent on the laser intensity. The threshold intensity for a single beamlet, where approximately 50% of the laser pulses successfully trigger an action potential, is ~4 µW/μm² (n=5 regions, 5 cells analyzed per region, performed in triplicates) at frequency of 1 Hz and pulse duration of 10 ms. We also demonstrate spatial summation of membrane potential in HL1-ChR2. Generated multiple foci can render the excited cells as individual inputs to elicit action potential in a syncytium. We demonstrate the dependence of the excitation probability on the foci gap distance. Two foci, each with sub-threshold intensity of 2 µW/μm² were generated. At spacing of 50 µm, the median excitation probability increases compared to single spot stimulation with the same intensity.

The tetrapyrrole chlorin e6 (Ce6) is actively used in photodynamic therapy (PDT) and as a test drug for sonodynamic therapy due to its ability to generate singlet oxygen. In this work, we have analyzed the absorption, photoluminescence (PL), and PL excitation spectra of Ce6 molecules in a nutrient medium and inside the melanoma cells. An analysis of the results shows that Ce6 inside the cancer cells with a low pH remains in monomeric form. A photodynamic test has also been conducted that confirmed the photodynamic effect produced by Ce6 in cancer cells by changing the signals from Ce6 and cells in the absorption and PL spectra.

Snapshot multispectral image sensors have been proposed as a key enabler for a plethora of multispectral imaging applications, from diagnostic medical imaging to remote sensing. With each application requiring a different set, and number, of spectral bands, the absence of a scalable, cost effective manufacturing solution of custom multispectral filter arrays (MSFAs) has prevented widespread MSI adoption. Despite recent nanophotonic-based efforts, such as plasmonic or high-index metasurface arrays, large-area MSFA manufacturing still consists of many-layer dielectric (Fabry Perot) stacks, requiring separate complex lithography steps for each spectral band and multiple material compositions for each. It is an expensive, cumbersome and inflexible undertaking, but yields optimal optical performance. Here, we demonstrate a manufacturing process that enables cost effective wafer-level fabrication of custom MSFAs in a single lithographic step, maintaining high efficiencies (~75%) and narrow linewidths (~25 nm) across the visible to near-infrared. By merging grayscale (analog) lithography with metal-insulator-metal (MIM) Fabry-Perot cavities, whereby exposure dose controls cavity thickness, we demonstrate simplified fabrication of MSFAs up to N-wavelength bands. The concept is first proven using low volume electron beam lithography, followed by the demonstration of large volume UV mask-based photolithography with MSFAs produced at the wafer-level. Our framework provides an attractive alternative to conventional MSFA manufacture, and metasurface-based spectral filters, by reducing both fabrication complexity and cost of these intricate optical devices, while increasing customizability.

In medical imaging, a number of imaging modalities have been used to visualize the structural information of different internal organs in the human body and some modalities can even visualize structures at a cellular level for diagnostic and treatment purposes. Optical resolution photoacoustic microscopy (OR-PAM) is one of the emerging imaging modalities to analyze the anatomy and functionality of tissues non-invasive. It is a hybrid imaging technology that combines photoacoustic (PA) contrast and acoustic resolution to reconstruct images of tissues in humans and animals. However, in OR-PAM the received ultrasonic signal by the thermal expansion of tissues has a low signal-to-noise ratio because most of the signal power is lost in the conversion process from light to acoustic waves which makes it difficult to visualize the structural information in the PA images. Traditional denoising methods such as wiener filter, bandpass filter, wavelet-based denoising, noise reduction by SVD and dictionary-based denoising methods can denoise PA images to some extent but it is still a difficult task to preserve structural information in the images by such methods. In this research, a convolutional autoencoder (CAE) based model is proposed to denoise and learn the structural patterns of blood vessels in PA images. To achieve this task, a CAE model is first trained between noisy and Gabor filtered sub-images, those contain the patterns of different vascular structures. Then, the trained model is used to approximate the denoise version of the input noisy sub-images of blood vessels. The proposed model is trained and tested on PA images of blood vessels of a mouse ear, acquired by the OR-PAM imaging system and the results show that our proposed method can effectively approximate and reconstruct the noisy vascular structures than traditional images denoising filtering methods.

The study aims at development and laboratory approbation of non-contact optical technique for early evaluation of microbial activity. Microorganisms’ activity is estimated by laser speckle contrast imaging technique in combination with image processing of obtained time varying speckle patterns. Laser speckle patterns were captured by CMOS sensor during illumination of growing bacteria colonies by low power (<30 mW, 635 nm) stabilized coherent light source. To validate proposed technique and image processing algorithm the vibrio natriegens bacteria are used. After analysis of several different experiments the following results were obtained: In the central part of the colony activity can be seen in 2.5-3 hours. Thus, earlier detection of bacterial activity is expected, i.e., earlier than 2-3 hours, which is much earlier than the standard counting methods used to count colony forming units (CFU).

The assessment of dental issues is done nowadays both clinically and radiologically. The latter includes radiographs that are based on X-ray radiation, i.e. intraoral radiography, panoramic radiography, and three-dimensional (3D) cone beam computed tomography (CBCT). In several cases, radiographs have limitations, as they do not reveal dental issues such as small cavities, enamel cracks, or tooth erosion. These aspects can be visible with another medical imaging technique, Optical Coherence Tomography (OCT). The aim of this study is to present a few results obtained with an in-house developed swept-source OCT (SS-OCT) system on several dental issues that cannot be visible on radiographs. These results prove that OCT can be utilized in dentistry, with advantages such as radiation free technique and superior resolution. This study presents both radiography and OCT images for different dental issues which include small cavities, metal crowns cracks, or crowns manufactured with different materials (i.e., zirconia, ceramics, or composite). Firstly, samples have been analyzed radiologically and some abnormalities could be detected, but they could be correctly assessed. Secondly, these abnormalities have been analyzed with the SS-OCT system and finally all images and collected data from both medical imaging techniques have been compared. One of the conclusions is that OCT is more appropriate than radiography for several dental issues such as those presented in this study. These two medical imaging techniques can therefore be complementary in dental medicine.

In an incoherent optical fiber bundle (IOFB), the spatial correspondence of its both ends is not given, which usually complicates its handling. There are, however, medical applications for which IOFBs are well suited: Ultra-high spatial resolution fiber spectroscopy, for example, requires densely packed thin fibers on one side of the bundle, while on the other bundle side a flexible arrangement of the fibers is necessary to prevent overlapping of the individual spectra. To make such IOFBs usable for information transmission, a calibration routine to match input and output of the bundle is necessary. The aim of the present work is to establish a calibration routine for densely packed IOFBs with a single fiber core diameter of 23 μm. A HeNe laser focused to illuminate only one single fiber at a time is forming the basis of the experimental setup. An image of the output side is taken, the respective brightness peak is detected and its coordinates are stored in a look-up table (LUT) to allow the fiber bundle input to be reconstructed from its signal output. Several validation and calibration steps have been established to ensure a reliable fiber assignment. In a test run, 94.3% of the 1,374 fibers of the bundle used for proof of concept could be assigned exactly and used for image transmission.

Numerical beam refocusing in OCT is used to increase lateral resolution in the out-of-focus areas for strongly focused beams. However, this approach is based on overlapping lateral scanning in the assumption of the same scatterers positions. In the presence of scatterer motions the numerical refocusing approach can fail. It limit the applicability of the numerical refocusing approach to such based on scatterer motion evaluation modalities as angiography and elastopgraphy. Due to hard controlling phantom experiments we evaluate the influence of motion of scatterers on numerical beam refocusing on the base of numerical simulated OCT scans. The motions of the scatterers are well controlled in the numerical model and its effect on the numerical refocusing approach can be quantified. For this study, a full-wave model for simulating images in spectral-domain optical coherence tomography (OCT) with rigorous accounting for the beam-focusing effects is used.

In this work, infrared (IR) experimental setup for exhaled breath analysis (BA-setup) and experimental setup for biological fluids analysis (FA-setup) based on quantum cascade laser was demonstrated. For breath analysis, we use the absorption spectroscopy with astigmatic multipass gas cells for biomarkers identification in human breath. The expected sensitivity of BA-setup was estimated. Mathematical methods for processing the human breath IR spectra were proposed. FA-setup uses spectral analysis of diffuse reflected radiation for identification of liquid and solid substances. Examples of the obtained experimental data were provided. Using FA-setup, several solid organic components and liquid substances were successfully indicated.

Raman micro-spectroscopy and different approaches for multivariate analysis were used for an investigation of subcellular regions of X-ray exposed single SH-SY5Y human neuroblastoma cells. Nucleus and cytoplasm regions of single cells were investigated after X-rays irradiation (0, 2, 4, 6 and 8 Gy). Cells fixed immediately after irradiation and 24h irradiation were considered. Principal component analysis (PCA) and interval-PCA (i-PCA) were used for analyzing the spectra in order to highlight the changes due to the different treatments. Biochemical changes occurring in the nucleus and cytoplasm regions of single cells upon X-ray irradiation were observed. The analysis of Raman spectra allowed us to detect modifications in the contribution from proteins, nucleic acids, lipids, and carbohydrates of cells, induced at different extent on the two cell regions. The biochemical changes occurring in these cells were also discussed by using an alternative approach, namely the analysis of difference spectra, obtained by subtracting the cytoplasm-related spectrum from the corresponding one detected at the nucleus. The proposed approach enabled us to evidence some features not outlined in previous investigations. The results showed that it is possible to study in a selective way the effects of ionizing radiation on different neuroblastoma cell spatial regions. An increase of the signal related to the nucleobases, a decrease of DNA and/or RNA backbone contribution, a protein rearrangement with changes in the secondary structure, and an increase in lipid saturation were observed. These results indicated that the development of accurate data analysis methods enabling to take into account the complexity of the Raman spectra of cells and tissues and the high number of spectra needed to consider the intrinsic variability of biological samples.

The recently designed Tpx3Cam camera based PLIM (Phosphorescence Lifetime IMaging) macro-imager was tested using an array of phosphorescent chemical and biological samples. A series of sensor materials prepared by incorporating the phosphorescent O2-sensitive dye, PtBP, into five polymers with different O2 permeability were imaged along with several commercial and non-commercial sensors based on PtBP and PtOEPK dyes. The PLIM images showed good lifetime contrast between the different materials, and phosphorescence lifetime values obtained were consistent with those measured by alternative methods. A panel of live tissues samples stained with PtBP based nanoparticle probe were also prepared and imaged under resting conditions and upon inhibition of respiration. The macro-imager showed promising results as a tool for PLIM of O2 in chemical and biological samples.

Breast cancer is a genetically heterogeneous disease characterized by various biomolecular and morphological features that affect the diagnosis, prognosis, and treatment response. In this study we combined cross-polarization optical coherence tomography (CP OCT) and multiphoton tomography (MPT), based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) to visualize tumor stroma and tumor cells in specimens of a human breast tissue. The data obtained by both techniques were compared with histopathology. The CP OCT and MPT images were quantitatively assessed to distinguish a breast normal tissue from a cancer as well as between a low and a high grade of cancer. Quantitative assessment of the CP OCT image included the calculation of signal attenuation coefficients separately for co- and cross- polarization channels and the formation the color-coded en-face distribution maps of these coefficients. The attenuation coefficient in cross- polarization channel showed better difference between breast cancer of low and high grades and distinguish them from normal tissue. The SHG images were processed using texture analysis techniques to quantify the density of collagen fibers in normal tissue and tumor. Thus, both imaging techniques have great potential to distinguish nontumorous and tumorous human breast tissue of varying degrees of malignancy and could provide identifying breast cancer margins for in surgery.

The major etiologies of gout and kidney stone formation are similar－hyperuremia , which is caused by unbalanced uric acid production/exertion. And gout is one of the important risk factor that promotes kidney stone formation. It’s worth noting that crystals retension/aggregation is the early stage of stone formation. However, the mechanism between gout and crystalluria remains unclear. Our previous studies found good correlation between auto-fluorescent crystals and urolithiasis. Higher proportion of auto-fluorescent crystalluria in gout patients were also noted. It this paper, we demonstrated characterization of auto-fluorescent urinary crystals in gout patients by micro-Raman system and laser confocal microscopy. Our purpose is to figure out the mechanism among gout flare, urolithiasis and auto-fluorescent crystal retention. And the results supported that fluorophore inside the crystals may be a powerful indicator for crystals retention. Further studies are required to validate our hypothesis.

Due to circumstances beyond the presenter's control, and audio recording was not possible for this presentation.

In the work, the effective diffusion coefficient of an aqueous solution of the pharmaceutical preparation rivanol in slices of human tooth dentin was experimentally determined using the method of reflection spectroscopy. Within the framework of the free diffusion model, in calculations using the second Fick law and the Bouguer-Lambert-Beer law for the optical model, the effective diffusion coefficient of the photosensitizer was calculated, which turned out to be equal on average (2.27 ± 0.32)·10^–6 cm²/s (n = 10).

Natural carbon isotopes (carbon-12 (¹²C) and carbon-13 (¹³C)) contained in human respiration can be used to detect various diseases (cachexia, helicobacter pylori). Isotope mass spectrometry (IRMS), which is widely used to determine carbon isotopes in human respiration has a high level of accuracy and sensitivity, but is a very complex and expensive technique. There is a less expensive way to detect carbon isotopes using an isotope-selective non-dispersive infrared spectrometer (NDIRS), but it is only suitable for simple breath tests when a small number of samples is required. Raman spectroscopy is well suited for the simultaneous detection of various gases in the analysis of human respiration, but the Raman signal from the carbon isotopes has a very low intensity, what makes their detection difficult. In this work, we demonstrate an effective system for detecting carbon isotopes ¹²CO₂ and ¹³CO₂ in human breath with an extremely low concentration level of ~ 0.01%. The Raman detector consists of a 5 W CW narrow-linewidth single-frequency solid-state laser at 532 nm, a focusing system with compensation for a spherical aberration, a gas cell which can withstand pressures up to 100 atmospheres and a high-resolution Czerny-Turner based spectrometer with a matrix, cooled by a Peltier element down to -40 °C. Such a system has a lower cost in comparison with analogues and can be used the medical diagnosis of various diseases.

Monitoring of person microcirculatory bed state is one of the important problems of modern medical diagnostics. Due to the fact that many diseases cause changes of microcirculatory blood flow velocity, and timely diagnosis of these diseases prevents the development of pathologies. Currently, there are many methods for assessing of the capillary bed state. However, the most effective diagnostic methods for determining the main parameters of microcirculation include the method of dynamic light scattering. In this paper the speckle correlation sensor of microcirculatory blood flow velocity registration is considered. Using this setup, monitoring of blood flow velocity of a group of people under various conditions were studied. The results of these studies are presented. The measurement results showed that the installation is sensitive to external factors and allows you to record rapid changes in the speed of capillary blood flow. Also, the rate of blood flow in patients varies, depending on the state of their microvasculature, this fact will allow doctors to monitor the state of the human capillary system.

The change of corneal shape in keratoconus subjects can impact the optical quality of the eye on the retina and it reduces the contrast sensitivity by light scattering. The aim of our study was to estimate the keratoconus subjects’ contrast sensitivity and visual acuity depending on keratoconus apex position. We included 45 keratoconus subjects (77 eyes), which have keratoconus in the first to the third stage, in our study. There were 33 eyes with keratoconus apex in the central part of the cornea and 46 eyes with keratoconus apex in the periphery of the cornea. Contrast sensitivity and visual acuity were measured at 3 m with and without the best possible spectacle correction using the FrACT program 3.9.3. The contrast sensitivity was measured at following frequencies – 1, 3, 5, 7, 9, 11, 13, and 15 cpd. The results showed that keratoconus subjects have lower contrast sensitivity in all spatial frequencies than subjects without pathological changes. The lowest contrast sensitivity was in keratoconus subjects with a central apex position compared to contrast sensitivity with peripheral apex position. The difference of contrast sensitivity between subjects with and without pathology increased up to 11 cpd but remained rather constant at the highest spatial frequencies – 11, 13, and 15 cpd. There was statistically significant difference between the median of visual acuity in subjects with central apex (0.42 decimal system) and subjects with peripheral apex (0.60 decimal system).