The Imaging Core will provide both fluorescence light microscopy and electron microscopy resources to Center Investigators to enable evaluation of muscle morphology and structural organization, and to localize specific molecular components in the sarcomeres, cytoskeleton, subcellular organelles and membranes. The broad long-term objective of this Core is to provide quantitative structural information that will be an essential link in the proposed Center's long-term goal to achieve a comprehensive understanding of multi-scale structure-function relationships in skeletal muscle. The Imaging Core will interface with the Phenotyping Core and the High-throughput Cell Analysis Core by assisting investigators in fluorescence light microscopy and transmission electron microscopy analyses of skeletal muscle tissues and cells isolated from wild-type and transgenic mice, or from healthy and diseased human muscles. The Core will provide Center investigators with training and assistance in the complex imaging technologies of Transmission Electron Microscopy and Confocal Laser Scanning Fluorescence Microscopy on fixed specimens, Confocal Spinning Disc and Wide-Field Fluorescence Microscopy of molecular dynamics in living cells, and Single-Molecule Fluorescence imaging using Total Internal Reflection Fluorescence (TIRF) Microscopy. The Core will provide training and assistance with routine aspects of sample preparation for microscopy, and training and access to microscopes in The Scripps Research Institute (TSRI) Microscopy Facility, and in the Center for Integrated Molecular Biosciences (CIMBio) Fluorescence Microscopy Suite at TSRI. The Core will also assist investigators in application of image analysis software to their experimental problems and in quantitative interpretation of fluorescent image data.
The Specific Aims are: 1) To provide training and technical assistance in skeletal muscle cell and tissue fixation and processing in preparation for fluorescence light microscopy and/or transmission electron microscopy;2) To provide training and access to fluorescence and electron microscopes at TSRI;3) To provide training and assistance in quantitative 2D and 3D image analysis software packages (Metamorph, Volocity Suite), and custom computational approaches to measure myofibril structure (Distributed Deconvolution);4) To upgrade the TIRF microscope to perform superresolution Photoactivation Localization Microscopy (PALM) and Stochastic Optical Resconstruction Microscopy (STORM), enabling nano-scale single-molecule fluorescence imaging in muscle cells and isolated myofibrils.
Muscle contraction relies on movements of repeating structures called sarcomeres, which are attached to the muscle membranes and tendons to generate force. Muscle diseases, such as myopathies and dystrophies exhibit disruptions in sarcomeres and membrane attachments, as well as alterations in muscle fiber structure due to degeneration and regeneration. Microscopic visualization of muscle structures will explain altered function, provide insight into causes of human myopathies and lead to better treatments.
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