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.

The thesis deals with LSCI (Laser Speckle Contrast Imaging), an optical method utilizing laser speckle contrast for the estimation of blood flow changes. LSCI is non-invasive and technically not demanding approach, capabilities of which have not yet been fully exploited. The literature review part contains detailed description of the operating principle, imaging techniques, potential for medical applications with considering the limiting factors. The main aim of the thesis is to design and construct a complete LSCI system including appropriate phantoms able to simulate blood flow through the tissue. Imaging algorithms for the obtained data evaluation were implemented in Matlab® development enviroment. Finally, the created system was tested using different acquisition parameters as well as varying the image processing schemes. The resulting qualitative flow images were subsequently discussed and confronted with the theoretical assumptions.

This thesis deals with introduction to optical method called laser speckle contrast imaging (LSCI). The thesis contains a description of the basic theory that is related to the method, the LSCI own principle, the characteristics of this technique, its applications and also a description of various variants of LSCI. The second part of the thesis deals with the modification of acquisition software to be suitable for measurement of multi-exposure LSCI (MESI), verification of this method by experiment and conclusions of evaluated of data.

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