A subsarcolemmal tubular system network (SSTN) has been detected in skeletal muscle fibers by confocal imaging after the removal of the sarcolemma. Here we confirm the existence and resolve the fine architecture and the localization of the SSTN at an unprecedented level of detail by examining extracellularly applied tubular system markers in skeletal muscle fiber preparations with a combination of three imaging modalities: confocal fluorescence microscopy, direct stochastic optical reconstruction microscopy, and tomographic electron microscopy. Three-dimensional reconstructions showed that the SSTN was a dense two-dimensional network within the subsarcolemmal space around the fiber, running ~500-600 nm underneath and parallel to the sarcolemma. The SSTN is composed of tubules ~95 nm in width with ~60% of the tubules directed transversely and >30% directed longitudinally. The deeper regular transverse tubules located at each A-I boundary of the sarcomeres branched from the SSTN, indicating individual transverse tubules that form triads are continuous with, but do not directly contact the sarcolemma. This suggests that the SSTN plays an important role in affecting the exchange of deeper tubule lumina with the extracellular space.
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