Cell transplantation and stem cell therapy are promising approaches for regenerative medicine and are of interest to researchers and clinicians worldwide. However, currently, no imaging technique that allows three-dimensional in vivo inspection of therapeutically administered cells in host tissues is available. Therefore, we investigate magnetomotive optical coherence tomography (MM-OCT) of cells labeled with magnetic particles as a potential noninvasive cell tracking method. We develop magnetomotive imaging of mesenchymal stem cells for future cell therapy monitoring. Cells were labeled with fluorescent iron oxide nanoparticles, embedded in tissue-mimicking agar scaffolds, and imaged using a microscope setup with an integrated MM-OCT probe. Magnetic particle-induced motion in response to a pulsed magnetic field of 0.2 T was successfully detected by OCT speckle variance analysis, and cross-sectional and volumetric OCT scans with highlighted labeled cells were obtained. In parallel, fluorescence microscopy and laser speckle reflectometry were applied as two-dimensional reference modalities to image particle distribution and magnetically induced motion inside the sample, respectively. All three optical imaging modalities were in good agreement with each other. Thus, magnetomotive imaging using iron oxide nanoparticles as cellular contrast agents is a potential technique for enhanced visualization of selected cells in OCT.

Laser speckle contrast imaging (LSCI) and its extension, multi-exposure laser speckle contrast imaging (MELSCI) are non-invasive techniques to monitor peripheral blood perfusion. One of the main drawbacks of LSCI and MELSCI in clinical use is that the techniques are sensitive to tissue movement. Moreover, the image background contributes to unnecessary data. The aim of this project was to develop and evaluate different methods to detect local motion artifacts and image backgrounds in LSCI and MELSCI. In this project, three different methods were developed: one using statistical analysis and two using machine learning. The method based on classical statistics was developed in MATLAB with a dataset made up of 1797 frames of 256 x 320 images taken from a recording of a hand where the thumb and middle finger were taking turns making small movements while the middle finger was the subject of three different states made by an occlusion cuff (baseline, occlusion, and reperfusion). The main filter that was used in the first method was the Hampel filter. Furthermore, networks for the machine learning method were developed in Python using the same dataset but with 20,000 small patches extracted from the dataset of sizes 3 x 3 to 21 x 21 pixels. The first machine learning method was based on two-dimensional data patches, hence no time dimension was included, while the second machine learning method used three-dimensional data patches where the time dimension was included (from 1s to 10s). The generation of ground truth for the dataset was manually created frame by frame in a ground truth generation graphical user interface (GUI) in MATLAB. To assess the three methods, the Dice coefficient was used. The statistical method resulted in a Dice coefficient of 0.7557. The highest Dice coefficient for the machine learning method with a 2D dataset was 0.2902 (patch size 13 x 13) and the lowest was 0.2372 (patch size 7 x 7). For the machine learning method with 3D datasets, the patch size of 21 x 21 x 4 resulted in the highest Dice coefficient (0.5173), and the 21 x 21 x 40 model had the lowest Dice coefficient (0.1782). Since the two methods based on temporal data proved to be performing best in this project, one conclusion for further development of an improved model is the usage of temporal data in the training of a model. However, one important difference between the statistical method and the three-dimensional machine learning method is that the statistical method does not handle fast perfusion changes as well as the machine learning method and can not detect image background and static tissue. Therefore, the overall most useful method to further develop is the three-dimensional machine learning method.

This thesis presents fundamental research in the field of laser vibrometry for the application to vibration measurements. A key concern for laser vibrometry is the effect of laser speckle which appears when a coherent laser beam scatters from an optically rough surface. The laser vibrometer is sensitive to changes in laser speckle which result from surface motions not in the direction of the incident beam. This adds speckle noise to the vibrometer output which can be indistinguishable from the genuine surface vibrations. This has been termed ‘pseudo-vibration' and requires careful data interpretation by the vibration engineer. This research has discovered that measurements from smooth surfaces, even when no identifiable speckle pattern is generated, can produce noise and therefore reference to speckle noise, in such circumstances, is inappropriate. This thesis has, therefore, adopted the more general term of pseudo-vibration to include noise generated from any surface roughness or treatment, i.e. including but not limited to speckle noise. This thesis develops and implements novel experimental methods to quantify pseudovibration sensitivities (transverse, tilt and rotation sensitivity) with attention focussed on commercially available laser vibrometers and consideration is given to a range of surface roughnesses and treatments. It investigates, experimentally, the fundamental behaviour of speckles and attempts to formulate, for the first time, a relationship between changes in intensity to pseudo-vibration sensitivity levels. The thesis also develops and implements models for computational simulation of pseudo-vibrations using the fundamental behaviour of speckles. The combination of experimentation and simulation improves current understanding of the pseudo-vibration mechanisms and provides the vibration engineer with a valuable resource to improve data interpretation. Two experimental methods of quantifying pseudo-vibration sensitivity are developed and successfully applied in the evaluation of transverse, tilt and rotation sensitivity for two models of commercial laser vibrometer. These evaluations cover both single beam (translational vibration measurement) and parallel beam (for angular vibration measurement) modes. The first method presented requires correction of the vibrometer measurement with an independent measurement of genuine velocity to produce an iii apparent velocity dominated by the required noise components. The second method requires a differential measurement using two vibrometers to cancel common components such as genuine velocity, leaving only uncorrelated noise from each measurement in the resulting apparent velocity. In each case, a third measurement is required of the surface motion component causing pseudo-vibration and this is used to normalise the apparent velocity. Pseudo-vibration sensitivity is then presented as a map showing the spectral shape of the noise, as a mean and standard deviation of harmonic peaks in the map and as a total rms level across a defined bandwidth. The simulations employ a novel and effective approach to modelling speckle evolution. Transverse and tilt sensitivity are predicted for the first time and are verified by the experimental study. They provide the vibration engineer with the potential to estimate pseudo-vibrations using a simple piece of software. The laser beam spot diameter has a large influence on the pseudo-vibration sensitivity. Transverse sensitivity has been quantified as around 0.03% and 0.01% (per order) of the transverse velocity of the surface for beam spot diameters of 100 μm and 600 μm respectively. Larger beam spots have been shown to significantly reduce transverse sensitivity and measurements from smoother surfaces have also shown a reduced level of transverse sensitivity. Tilt sensitivity has been quantified at about 0.1 μms-1/degs-1 and 0.3 μms-1/degs-1 (per order) of angular velocity of the surface for beam spot diameters of 100 μm and 600 μm respectively. Smaller beam spot diameters significantly reduce tilt sensitivity. The surface roughness or treatment has been shown to have little effect on the level of tilt sensitivity. Rotation sensitivity has been quantified at approximately 0.6 μms- 1/rads-1 and 1.9μms-1/rads-1 (per order) of rotation velocity of the rotor for 90 μm and 520 μm. Smaller beam spot diameters have shown a significant reduction in rotation sensitivity and measurements on smoother surfaces have shown a reduced rotation sensitivity. Focussing the laser beam approximately on the rotation axis has also shown a significant reduction in rotation sensitivity. Parallel beam rotation sensitivity has been quantified at 0.016 degs-1/rads-1 and it is demonstrated that this can adequately be estimated using the single beam rotation sensitivity.

La diffusion électromagnétique par des surfaces rugueuses concerne un ensemble très vaste de problèmes actuels en optronique (maîtrise des signatures/cloaking, analyse des signatures laser et infrarouge, imagerie active, localisation de la lumière, imagerie optique haute résolution, modélisation des interactions lumière matière et des signatures optiques, applications photovoltaïques et détecteurs infrarouges, biotechnologie). Les travaux réalisés dans le cadre de la thèse "Contrôle de la lumière par des éléments de surface désordonnés'' consistent à réaliser expérimentalement des surfaces rugueuses dont les propriétés statistiques sont contrôlées. Pour cela, un banc expérimental de photofabrication a été développé, utilisant sles propriétés statistiques des figures de speckle issues d'un faisceau laser mis en forme spatialement.Les surfaces réalisées présentent ainsi des propriétés statistiques qui n'existent pas à l'état naturel (fonction d'autocorrélation non gaussienne). Au delà de ces surfaces photofabriquées, le travaux de cette thèse s'intéressent également aux propriétés de surfaces de silicium fortement rugueuses caractérisées de Black Silicon. / Scattering of electromagnetic waves from rough surfaces is involved in a wide area of research in optronics (cloaking, laser and infrared signature analysis, active imaging, light localisation, high resolution optical imaging, laser-matter interaction and optical signature modelling, photovoltaics, infrared sensors, biotechnologies). Studies performed during the thesis ``Ligth control by random surface elements'' consist in the experimental fabrication of rough surfaces which statistical properties are controled. An experimental setup has been implemented, using the properties of a spatially shaped laser speckle pattern. The photofabricated surfaces show statistical properties that do not exist in nature such as non Gaussian autocorrelation function. Beyond these photofabricated surfaces, we also studied very rough surfaces of semi-conductor known as Black Silicon.

Esse trabalho apresenta o método de correlação angular de speckle. Esse método foi melhorado para ser aplicado na medição de rugosidade na faixa de 30 m a 0,1 m. Um programa de computador foi desenvolvido para processar imagens e calcular a correlação angular, e novas fórmulas matemáticas foram deduzidas. / This work presents the speckle pattem angular correlation method. This method was improving to be applicable in the roughness measuring in the range of 0,1 m to 30 m. A software was developed to process image and to calculate the angular correlation, and new mathematical equations were formulating.

This dissertation presents the development and use of a novel optical imaging system capable of monitoring changes in blood flow, oxygenated hemoglobin, deoxygenated hemoglobin, and absolute pO₂ in the brain. There are several imaging modalities capable of monitoring these parameters separately. Laser speckle contrast imaging (LSCI) and multi-spectral reflectance imaging (MSRI) have been used to monitor relative blood flow and hemoglobin changes respectively. Phosphorescence quenching, while not typically used for imaging, is capable of noninvasive measurements of pO₂. Combining these three techniques has led to the development of an imaging system that could ultimately lead to a better understanding of brain physiology. By combining techniques such as LSCI and MSRI, it becomes possible to estimate the cerebral metabolic rate of oxygen (CMRO₂), an important indicator of neuronal function. It is equally important to understand absolute pO₂ levels so that oxygen metabolism can be examined in context. Integrating phosphorescence quenching and a spatial light modulator into the imaging system allowed absolute pO₂ to be simultaneously measured in distinct regions. This new combined system was used to investigate pathophysiological conditions such as cortical spreading depression (CSD) and ischemia. The observed hemodynamic changes associated with these events were largely dictated by baseline oxygen levels and varied significantly in different regions. This finding highlighted the importance of having a system capable of monitoring hemodynamic changes and absolute pO₂ simultaneously while maintaining enough spatial resolution to distinguish the changes in different regions. It was found that animals with low baseline pO₂ were unable to deliver enough oxygen to the brain during events like CSD because of the high metabolic demand. In order for this technique to become more prevalent among researchers, it is essential to make it cost effective and simple to use. This was accomplished by replacing the expensive excitation sources with cheaper light emitting diodes (LEDs) and redesigning the software interface so that it was easier to control the entire device. The final system shows the potential to become a key tool for researchers studying the role of absolute pO₂ and other hemodynamic parameters during pathophysiological conditions such as CSD and ischemia. / text

There has been widespread interest in corneal biomechanics over recent years, driven largely by the advancements in, and the popularity of refractive surgery techniques and subsequent concerns over their safety. Lately there has been interest into whether crosslinking, which is currently used for the treatment of keratoconus, could be developed as a minimally invasive technique to change the refractive power of the cornea by selectively changing the corneal biomechanics in specific regions to induce a shape change. Successful application of this technique requires a detailed understanding of corneal biomechanics and so far, little is known about the biomechanics of this complex tissue. The current lack of understanding can be mostly attributed to the absence of a suitable measurement technique capable of examining the dynamic behaviour of the cornea under physiological loading conditions. This thesis describes the development of a novel full-field, ex vivo, measurement method incorporating speckle interferometric techniques, to examine the biomechanics of the cornea before and after crosslinking in response to hydrostatic pressure fluctuations representative of those that occur in vivo during the cardiac cycle. The eventual measurement system used for the experiments detailed in this thesis incorporated; an Electronic Speckle Pattern Interferometer (ESPI), a Lateral Shearing Interferometer (LSI) and a fringe projection shape measurement system. The combination of these systems enabled the 3-dimensional components of surface displacement and the 1st derivative of surface displacement to be determined in response to small pressure fluctuations up to 1 mmHg in magnitude. The use of both ESPI and LSI together also enabled the applicability of LSI for measurement of non-flat surfaces to be assessed, and limitations and error sources to be identified throughout this work. To enable the measurement of corneal biomechanics, part of this thesis was concerned with the design of a bespoke loading rig. A chamber was designed that could accommodate tissue of both porcine and human origin. This chamber was linked to a hydraulic loading rig, whereby the cornea could be held at a baseline pressure representative of normal intraocular pressure and small pressure variations could be introduced by the automated vertical movement of the reservoir supplying the chamber. Experiments were conducted on a range of non-biological samples with both flat and curved surface topography, and both uniform and non-uniform mechanical properties, to determine if the measurement configuration was giving the expected measurement data and the loading rig was stable and repeatable. Following experiments on non-biological samples, a range of experiments were conducted on porcine corneas to develop a suitable testing methodology and address some of the challenges associated with corneal measurement, including transparency and hydration instability. During these investigations, a suitable surface coating was identified to generate an adequate return signal from the corneal surface, while not interfering with the response. Alongside this, the natural variation in the response of the cornea was investigated over the total experimental time, and a range of data was presented on corneas before and after crosslinking, which confirmed the suitability of the measurement methods for the assessment of crosslinking. Ultimately, a small sample size of six human corneas were investigated before and after crosslinking in specific topographic locations. From the experiments on human and porcine corneas, full-field maps of surface deformation have been presented, and a compliant region incorporating the peripheral and limbal areas has been identified as being fundamental to the response of the cornea to small pressure fluctuations. In addition to this, the regional effects of crosslinking in four different topographic locations on corneal biomechanics have been evaluated. From this, it has been demonstrated that crosslinking in specific regions in isolation can influence the way the cornea deforms to physiological-scale fluctuations in hydrostatic pressure and this could have implications for refractive correction.

Esse trabalho apresenta o método de correlação angular de speckle. Esse método foi melhorado para ser aplicado na medição de rugosidade na faixa de 30 m a 0,1 m. Um programa de computador foi desenvolvido para processar imagens e calcular a correlação angular, e novas fórmulas matemáticas foram deduzidas. / This work presents the speckle pattem angular correlation method. This method was improving to be applicable in the roughness measuring in the range of 0,1 m to 30 m. A software was developed to process image and to calculate the angular correlation, and new mathematical equations were formulating.

Le vieillissement est un facteur de risque des maladies cardiovasculaires. Il est associé à des altérations fonctionnelles et structurelles du système vasculaire.Une étude approfondie du processus de vieillissement et le développement de systèmes d’imagerie et des traitements de données associés deviennent donc une priorité. Par l’analyse d’images de contraste par speckle laser (LSCI), l’objectif de cette thèse est d’étudier l’influence de l’âge sur la micro circulation.Pour ce faire, des données de LSCI ont été acquises sur l’avant-bras de sujets sains jeunes et âgés. A partir de modèles mathématiques, nous avons déterminé la vitesse des érythrocytes de la micro circulation chez les deux groupes de sujets. Par ailleurs, nous avons également mené une étude de la complexité de séries temporelles d’ LSCI s’appuyant sur des mesures d’entropie multi échelle. Nos résultats montrent que : 1) le groupe de sujets plus âgés présente des valeurs de vitesse des globules rouges significativement plus élevées que celles des sujets jeunes à l’hyperémie réactive post-occlusive; 2) les fluctuations des séries temporelles de LSCI dans le groupe des sujets jeunes ont une complexité supérieure à celles du groupe de sujets âgés. Ces modifications observées sur la micro circulation pourraient être attribuées à des modifications du système vasculaire dans son ensemble. La compréhension de ces altérations pourrait conduire à de nouvelles perspectives en matière de prévention et de traitement des pathologies liées à l’âge. / Aging is a primary risk factor for cardiovascular diseases. It is associated with functional and structural alterations in the vascular system. Therefore, a deep study of the aging process and the development of imaging systems and associated processing become of the utmost importance. By processing laser speckle contrast images (LSCI), this PhD work aims at studying the influence of age on microcirculation. In our work, LSCI data were acquired from the skin forearm of healthy subjects, subdivided into two age groups (younger and older). From mathematical models, we determined red blood cells velocity in microcirculation in the two groups of subjects. Moreover, we applied multiscale entropy-based algorithms to LSCI time series in order to study the complexity of microvascular signals. Our main findings are: 1) the older group has significantly higher velocity values than the younger group at post-occlusive reactive hyperaemia; 2) LSCI fluctuations in the younger group have significantly higher complexity than those of the older group. Age-related changes in skin microcirculation can be attributed to alterations in the vascular system as a whole. Understanding these changes in the microcirculatory system may give new insights for prevention and treatment of age-related diseases

Microcirculation is defined as the blood flow in the smallest blood vessels. Laser speckle contrast imaging (LSCI) is a full field imaging technique that provides instantaneous 2-D perfusion maps of illuminated tissues based on speckle contrast. Perimed’s Perfusion Speckle Imager (PSI) is a medical device developed at Perimed AB that exploits LSCI to measure tissue blood perfusion. In this thesis work, a robust Autofocus (AF) algorithm for PSI was implemented. AF is a procedure to drive PSI camera to reach the depth of focus and acquire sharp images, that relies only on signal processing. Therefore, several Blind image sharpness assessment (BISA) methods, to judge the degree of image sharpness, were compared to choose which BISA method to incorporate in the algorithm. An optimized focus scanning technique was implemented to more efficiently find the depth of focus. When working with LSCI, speckle is a source of noise that destroys image content. Experiments were performed to study laser speckle filtration: digital filters were employed to attenuate the speckle noise that corrupted details in the acquired images. Finally, two methods to perform AF were provided. These procedures were proven practically with LED images. However, with laser source image information is corrupted by speckle despite the application of digital filters and AF remains a real challenge. Moreover, important hardware limitations require to be overcome to make the technique real-time. Focus motor speed should be higher to acquire images at different focus positions faster which could benefit the speed of the AF procedure and speckle filtration.