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Sarcomere length non-uniformities occur at all structural levels of skeletal muscles and have been associated with important mechanical properties. Changes in sarcomere length non-uniformities in the nano- and sub-nanometer range have been used to explain muscle properties and contractile mechanisms. Typically, these measurements rely on light microscopy with a limited spatial resolution. One critical aspect in sarcomere length determination is the relatively arbitrary choice of intensity thresholds used to delineate sarcomere structure