Abstract: Nonlinear multicontrast microscopy is established in this study as an important tool for understanding biological structure and function of muscle cells. Second harmonic generation, third harmonic generation and multi-photon excitation fluorescence are acquired simultaneously in order to establish the origin of nonlinear signal generation in myocytes, and investigate myocyte structure and functionality during muscle contraction. Using structural cross-correlation image analysis, an algorithm developed specifically for this research, for the first time, third harmonic generation is shown to originate from the mitochondria in myocytes. The second harmonic, which is generated from the anisotropic bands of the sarcomeres, is further shown to be dependent on the crystalline order of the sarcomeres, thereby providing a potential diagnostic tool to evaluate disorder in muscle cells. The combination of the second and third harmonic provides complementary information that can be used to further elucidate the basic principles of muscle contraction. Time-lapse nonlinear microscopic imaging showed structural and functional dynamics in the myocytes. The second harmonic contrast revealed nonsynchronized nanocontractions of sarcomeres in relaxed, non-contracting, cardiomyocytes and Drosophila muscle samples, providing insight into the asynchronous behaviour of individual sarcomeres. Furthermore, macrocontracting samples were found to exhibit a synchronization of nanocontractions, providing new evidence for how muscles contract. Dynamic image correlation analysis, another algorithm developed specifically for this investigation, is used to reveal networks of mitochondria, which show fluctuations of multi-photon excitation fluorescence and third harmonic generation signals. The intensity fluctuations in the networks reveal both slow and fast dynamics; phase shifts of the slow dynamics between different networks are observed. Fast dynamics appear only in the inner networks, suggesting functional difference between interfibrillar and subsarcolemma mitochondria. The groundwork for studying bioenergetics of mitochondria in cardiomyocytes with nonlinear multimodal microscopy is fully developed in this work. The origin of the nonlinear signals and the development of the image analysis techniques provide a solid foundation to further study of muscle contractility and bioenergetics.

Abstract: Nonlinear multicontrast microscopy is established in this study as an important tool for understanding biological structure and function of muscle cells. Second harmonic generation, third harmonic generation and multi-photon excitation fluorescence are acquired simultaneously in order to establish the origin of nonlinear signal generation in myocytes, and investigate myocyte structure and functionality during muscle contraction. Using structural cross-correlation image analysis, an algorithm developed specifically for this research, for the first time, third harmonic generation is shown to originate from the mitochondria in myocytes. The second harmonic, which is generated from the anisotropic bands of the sarcomeres, is further shown to be dependent on the crystalline order of the sarcomeres, thereby providing a potential diagnostic tool to evaluate disorder in muscle cells. The combination of the second and third harmonic provides complementary information that can be used to further elucidate the basic principles of muscle contraction. Time-lapse nonlinear microscopic imaging showed structural and functional dynamics in the myocytes. The second harmonic contrast revealed nonsynchronized nanocontractions of sarcomeres in relaxed, non-contracting, cardiomyocytes and Drosophila muscle samples, providing insight into the asynchronous behaviour of individual sarcomeres. Furthermore, macrocontracting samples were found to exhibit a synchronization of nanocontractions, providing new evidence for how muscles contract. Dynamic image correlation analysis, another algorithm developed specifically for this investigation, is used to reveal networks of mitochondria, which show fluctuations of multi-photon excitation fluorescence and third harmonic generation signals. The intensity fluctuations in the networks reveal both slow and fast dynamics; phase shifts of the slow dynamics between different networks are observed. Fast dynamics appear only in the inner networks, suggesting functional difference between interfibrillar and subsarcolemma mitochondria. The groundwork for studying bioenergetics of mitochondria in cardiomyocytes with nonlinear multimodal microscopy is fully developed in this work. The origin of the nonlinear signals and the development of the image analysis techniques provide a solid foundation to further study of muscle contractility and bioenergetics.

L'Imagerie par Résonance Magnétique (IRM) présente deux caractéristiques principales. La première, sa capacité à manipuler le contraste, constitue son principal avantage par rapport aux autres modalités d'imagerie. Cela permet d'obtenir des informations complémentaires pour une meilleure détectabilité et une meilleure précision dans le diagnostic. Cela est particulièrement appréciable pour les pathologies du myocarde. La seconde caractéristique de l'IRM est également l'un de ces principaux inconvénients : le processus d'acquisition est relativement lent. De ce fait, les mouvements du patient constituent un obstacle important puisqu'ils perturbent ce processus d'acquisition, ce qui se traduit par des artéfacts dans l'image reconstruite. L'imagerie cardiaque et abdominale sont donc particulièrement sensibles à cette problématique du mouvement. L'objectif de cette thèse est donc de proposer une méthode de correction de mouvement intégrable dans un contexte multi-contraste. Nous avons étudié dans un premier temps la question de la correction de mouvement seule. Pour cela, nous nous sommes plus particulièrement intéressés à la méthode GRICS déjà développée au laboratoire IADI. Cette méthode permet la reconstruction conjointe d'une image sans artéfact et d'un modèle de mouvement non rigide permettant de corriger les déplacements qui surviennent pendant l'acquisition. Le premier apport majeur de cette thèse a consisté à améliorer la méthode GRICS, notamment pour l'adapter à l'imagerie volumique 3D. Il s'agit d'une nouvelle méthode de régularisation adaptative particulièrement adaptée au problème inverse posé dans GRICS. Le second apport majeur de cette thèse a consisté à gérer la correction de mouvement GRICS de manière conjointe sur des acquisitions présentant des contrastes différents. Il s'agit de concevoir l'examen IRM comme un tout et d'exploiter au mieux les informations partagées entre les différents contrastes. Toutes ces méthodes ont été appliquées et validées par des simulations, des tests sur fantôme, sur volontaires sains et sur des patients dans la cadre d'études cliniques. L'application cardiaque a été particulièrement visée. Les méthodes développées ont permis d'améliorer le processus d'acquisition et de reconstruction dans le contexte clinique réel / Magnetic Resonance Imaging (MRI) has two main features. The first one, its ability to manipulate contrast, is a major advantage compared to the other imaging modalities. It allows to access complementary information for a better detectability and a diagnostic more accurate. This is especially useful for myocardium pathologies. The second feature of MRI is also one of its main drawbacks: the acquisition process is slow. Therefore, patient motion is a significant obstacle because it disturbs the acquisition process, which leads to artifacts in the reconstructed image. Cardiac and thoracic imaging are particularly sensitive to this motion issue. The aim of this thesis is to develop a new motion correction method that can be integrated in a multi-contrast workflow. In a first phase, we studied apart the motion correction problem. To do so, we focused more particularly on the GRICS method which was already developed in the IADI laboratory. This method allows the joint reconstruction of an image free from artifact and a non-rigid motion model that describes the displacements occurring during the acquisition. The first major contribution of this thesis is an improvement of the GRICS method consisting mainly in adapting it to the 3D imaging. This was achieved with a new adaptive regularization method that perfectly suits the inverse problem posed in GRICS. The second major contribution of this thesis consists in the simultaneous management of the motion correction on multiple acquisitions with different contrasts. To do so, the MRI examination is considered as a whole. Thus we make the most of information shared between the different contrasts. All these methods have been applied and validated by simulations, tests on phantom, on healthy volunteers and on patients as part of clinical studies. We aimed more particularly at cardiac MR. Finally the developed methods improve the acquisition and reconstruction workflow in the framework of a real clinical routine

Die vorliegende Arbeit präsentiert neuartige schnelle Bildgebungstechniken für die Hoch- und Ultrahochfeld Magnetresonanztomographie. Zunächst werden die Grundprinzipien schneller Spin-Echo Techniken beleuchtet. Diese physikalischen Überlegungen bilden die Grundlage für die Entwicklung modifizierter Techniken. In einer ersten Entwicklungsstufe wird eine neue Variante der schnellen Spin-Echo Bildgebung vorgestellt. Diese Technik generiert anatomischen und funktionellen Bildkontrast innerhalb von nur einer Datenaufnahme. Der entscheidende Vorteil des entwickelten Ansatzes besteht in einer wesentlichen Verkürzung der Messzeit. Darüber hinaus wird eine deutliche Reduktion von Bildfehlern ermöglicht, die im konventionellen Fall häufig durch Bewegung erzeugt werden. Die zweite Entwicklungsstufe befasst sich mit der Implementierung einer schnellen Spin-Echo Technik zur Abbildung des physikalischen Phänomens der Brownschen Molekularbewegung. Diffusionsmessungen der Molekülbewegungen sind durch die Überlagerung von makroskopischen Bewegungen sehr anspruchsvoll. Diese Schwierigkeit wird in der vorliegenden Arbeit methodisch überwunden, indem eine diffusionsgewichtete schnelle Spin Echo Technik implementiert wird. Die dritte Entwicklungsstufe konzentriert sich auf suszeptibiltätsgewichtete schnelle Spin-Echo Bildgebung. Herkömmliche Techniken zur suszeptibiltätsgewichteten Bildgebung sind anfällig für Artefakte, die sich in Signalauslöschungen äußern. Um dieser Herausforderung methodisch zu begegnen, untersucht diese Arbeit das Potential einer suszeptibiltätsgewichteten schnellen Spin-Echo Technik zur Charakterisierung der Mikrostruktur des Herzmuskels bei 7.0 T. Ein Ziel der in dieser Arbeit neu entwickelten schnellen Spin-Echo Methoden besteht darin, Limitierungen bestehender Techniken zu beheben. Damit soll richtungsweisend über die Grundlagenforschung hinaus die Basis für klinische Anwendungen der entwickelten physikalischen Erkenntnisse und Methoden gelegt werden. / This thesis presents novel fast imaging techniques for magnetic resonance imaging. Rapid Acquisition with Relaxation Enhancement (RARE) is a fast imaging technique. An ever growing number of clinical applications render clinically and physically motivated advancement of RARE imaging necessary. This thesis focuses on the advancement of RARE imaging. For this purpose, the basic principle of RARE imaging is examined. The first part proposes a novel RARE variant which provides simultaneous anatomical and functional contrast within one acquisition. This approach provides an alternative versus conventional RARE variants where sequential acquisitions are put to use to achieve different image contrasts. With the speed gain of the proposed approach a substantial shortening of scanning time can be accomplished together with a reduction in the propensity for motion. The second part focuses on diffusion weighted MRI. Probing diffusion on a micrometer scale is challenging because of MRI’s sensitivity to bulk motion. Unfortunately, conventional rapid diffusion weighted imaging techniques are prone to severe image distortions. Realizing this constraint, a diffusion weighted RARE technique that affords the generation of diffusion weighted images free of distortion is implemented. The third part is formed around susceptibility weighted MRI. The underlying biophysical mechanisms allow the assessment of tissue microstructure. Common susceptibility weighted imaging techniques are prone to image artifacts. Recognizing the opportunities of susceptibility weighted MRI the potential of a susceptibility weighted RARE technique is investigated with the goal to assess myocardial microstructure. The goal of the novel RARE developments is to overcome constraints of existing imaging techniques. The physical considerations and the novel methodology introduced in this thesis are brought beyond the scope of basic research. Moreover, the foundation for clinical applicability is created.