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.