Laser speckle contrast imaging (LSCI) is a non-invasive method for assessment of microcirculatory blood flow. The technique is based on analysis of speckle patterns to build 2D maps of perfusion with high spatial and temporal resolution. A drawback of the method is that it is highly sensitive to motion artifacts since the perfusion estimates are based on quantification of the motion blurring in the images. The camera is today limited to a bulky stand for good measurements, but even as it is fixed, it does not ensure that the patient is completely still. In many clinical settings, it would be advantageous to have a more flexible camera and to be able to detect if an image is influenced by external motion. Multi-exposure laser speckle contrast imaging (MELSCI) is an extension to LSCI that utilizes the contrast from multiple exposure times. The gain in information has paved way for more accurate perfusion estimates. The technique has been limited due to its computational complexity, but recently a real time system has been developed. The goals of this thesis was twofold, firstly find a quantifiable measure of motion artifacts to be able to evaluate and compare LSCI and MELSCI. Secondly, propose an algorithm that detects movements in LSCI recordings. Motion artifacts in LSCI and MELSCI were investigated by developing a setup where repeatable movements could be made. Measurements of a hand influenced by motions of different speeds and directions were acquired and the relative difference between motion and static states were calculated and compared for the two systems. The relative difference of the MELSCI measurements were lower for all speeds above 0.57 mm/s, indicating more robustness to motion artifacts. A detection algorithm using image registration to calculate the instantaneous speed in each frame of the recording was developed. The method successfully detects movements perpendicular to the camera and shows that the intensity images of an LSCI recording can be used to give a direct indication of when movement has occurred.

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 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

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.

Cerebral blood flow (CBF) measures are central to the investigation of ischemic strokes, spreading depressions, functional and neuronal activation. Laser Speckle Contrast Imaging (LSCI) is an optical imaging technique that has been used to obtain CBF measures in vivo at high spatial and temporal resolutions, by quantifying the localized spatial blurring of backscattered coherent light induced by blood flow. Despite being widely used for biomedical applications, LSCI's critical limitations such as its tendency to underestimate large flow changes and its inability to accurately estimate CBF through a thinned skull have not been overcome. This dissertation presents a new Multi Exposure Speckle Imaging (MESI) technique that combines a new instrument and mathematical model to overcome these limitations. Additionally, in a pilot clinical study, an adapted neurosurgical microscope was used to obtain intra-operative LSCI images of CBF in humans. The MESI instrument accurately estimates experimental constants by imaging backscattered speckles over a wide range of the camera's exposure durations. The MESI mathematical model helps account for light that has scattered from both static and moving particles. In controlled flow experiments using tissue simulating phantoms, the MESI technique was found to estimate large changes in flow accurately and the estimates of flow changes were found to be unaffected by the presence of static particles in these phantoms. In an in vivo experiment in which the middle cerebral artery in mice was occluded to induce ~100% reduction in CBF, not only was the reduction in CBF accurately estimated by the MESI technique but these estimates of CBF changes were found to be unaffected by the presence of a thinned skull. The validity of statistical models used to derive the MESI mathematical model was confirmed using in vivo dynamic light scattering (DLS) measurements of CBF in mice. The MESI technique's potential to estimate absolute values of CBF in vivo was demonstrated by comparing CBF estimates obtained using the MESI technique to DLS measurements. The MESI technique's ability to measure CBF changes quantitatively through a thinned skull makes it particularly useful in chronic and long term studies leading to the development of better, more accurate stroke models. / text

La microcirculation cutanée a été proposée comme modèle d’étude de la dysfonction microvasculaire globale dans les maladies cardiovasculaires. Par ailleurs, elle est spécifiquement atteinte dans la sclérodermie systémique (SSc), qui est une maladie dysimmunitaire rare, particulièrement invalidante, caractérisée par une fibrose cutanée et viscérale associée à une atteinte microvasculaire diffuse et la présence d’auto anticorps dirigés contre des antigènes cellulaires. L’exploration de la fonction microvasculaire cutanée suscite donc un réel intérêt, même s’il n’existe pas de technique standardisée pour l’étude de la fonction microvasculaire, en particulier endothéliale.La première partie de ce travail a porté sur l’étude physiologique de la microcirculation cutanée chez le volontaire sain, en utilisant les méthodes les plus récentes adaptées à l’étude fonctionnelle de la microcirculation (tests de réactivité vasculaire couplés à l’enregistrement du flux sanguin cutané par laser speckle contrast imaging). Dans une seconde partie, nous avons étudié la pathologie de la microcirculation cutanée dans la sclérodermie systémique, en utilisant les mêmes d’étude fonctionnelle de la microcirculation. La dernière partie de cette thèse a été consacrée à l’étude d’une nouvelle approche pharmacologique et thérapeutique dans la prise en charge des manifestations vasculaires cutanées périphériques identifiées chez les patients. Nous avons évalué l’effet vasodilatateur du tréprostinil, analogue de la prostacycline, sur le flux sanguin cutané de divers zones anatomiques, chez le volontaire sain, le patient atteint de SSc, le patient diabétique et lors d’un refroidissement local dans la SSc. / Cutaneous microcirculation has been proposed as a model to study the global microvascular dysfunction occurring in cardiovascular diseases. Furthermore, it is specifically impaired in systemic sclerosis (SSc), which is a rare and particularly invalidating auto-immune disease, characterized by a cutaneous and visceral fibrosis, associated with a diffuse microvascular impairment and auto-antibodies targeting some cellular antigens. The study of cutaneous microvascular function provides a real interest despite the lack of available standardized techniques, particularly to explore endothelial microvascular function.In the first part of this work, we aimed to study the physiology of cutaneous microcirculation in healthy volunteers, using the more recent methods in this field, adapted to functional study of microcirculation (vascular reactivity tests coupled with cutaneous blood flow recording by laser speckle contrast imaging). The second part of our work aimed to study the pathology of cutaneous microcirculation in SSc volunteers, by using the same functional exploration methods. The last part of this work has been dedicated to a new pharmacologic and therapeutic approach for the management of peripheral cutaneous vascular manifestations in patients, using innovating technics as cutaneous iontophoresis. We studied the vasodilator effect of treprostinil, a prostacycline analogue, on cutaneous blood flow in several anatomic regions in healthy subject, SSc patient and diabetic patient, and also during a local cooling in SSc.

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