Key result
Minute variations in A-band threshold choice resulted in average sarcomere and half-sarcomere length changes of 28 nm and 18 nm, respectively, demonstrating that sub-nanometer measurements using light microscopy are unreliable noise.
Why the study?
Sarcomere length measurements rely on arbitrary intensity thresholds in light microscopy, which may cause centroid shifts and alter measured sarcomere lengths.
Sarcomere length measurements in the nanometer and sub-nanometer range obtained with light microscopy represent noise, suggesting a functional resolution limit of 50-100 nm is more appropriate.
Sub-nm sarcomere lengths by light microscopy warrant skepticism; leaves open reliable nanoscale methods for cardiac muscle research.
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 structures, such as A-bands or Z-lines. In experiments, these structures are typically distorted, intensity profiles vary, and baselines drift, resulting in asymmetric intensity patterns, causing changes in the centroid location of these structures depending on threshold choice, resulting in changes of sarcomere lengths. The purpose of this study was to determine the changes in (half-) sarcomere lengths associated with small changes in the A-band threshold choice. Sarcomere and half-sarcomere length changes for minute variations in A-band threshold were 28 nm (±28 nm) and 18 nm (±22 nm), respectively, and for the entire feasible range of thresholds across A-bands were 123 nm (±88 nm) and 99 nm (±105 nm), respectively. We conclude from these results that (half-) sarcomere lengths in the nanometer range obtained with light microcopy are noise, and the functional implications associated with such data should be discarded. We suggest that a functional resolution for sarcomere length of 100 nm (0.1 µm) is reasonable and 50 nm (0.05 µm) might be possible under ideal conditions.
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Schmidt et al. (2021) studied this question. A-band threshold variation was evaluated on Change in sarcomere and half-sarcomere lengths for minute variations in A-band threshold (28 nm (± 28 nm) for sarcomere length). Minute variations in A-band threshold choice resulted in average sarcomere and half-sarcomere length changes of 28 nm and 18 nm, respectively, demonstrating that sub-nanometer measurements using light microscopy are unreliable noise.
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