In Vivo Sarcomere Length Measurement in Whole Muscles during Passive Stretch and Twitch Contractions.

Muscle force is dictated by micrometer-scale contractile machines called sarcomeres. Whole-muscle force drops from peak force production to zero with just a few micrometers of sarcomere length change. No current technology is able to capture adequate dynamic sarcomere data in vivo, and thus we lack fundamental data needed to understand human movement and movement disorders. Methods such as diffraction, endoscopy, and optical coherence tomography have been applied to muscle but are prohibitively invasive, sensitive to motion artifact, and/or imprecise. Here, we report dynamic sarcomere length measurement in vivo using a combination of our recently validated resonant reflection spectroscopy method combined with optical frequency domain interferometry. Using a 250-µm-wide fiber optic probe, we captured nanometer sarcomere length changes from thousands of sarcomeres on the sub-millisecond timescale during whole-muscle stretch and twitch contraction. We believe that this demonstrates the first large-scale sensing of sarcomere dynamics in vivo, which is a necessary first step to understand movement disorders and to create patient-specific surgical interventions and rehabilitation.

Quantification of sarcomere length distribution in whole muscle frozen sections.

Laser diffraction (LD) is a valuable tool for measuring sarcomere length (Ls), a major determinant of muscle function. However, this method relies on few measurements per sample that are often extrapolated to whole muscle properties. Currently it is not possible to measure Ls throughout an entire muscle and determine how Ls varies at this scale. To address this issue, we developed an actuated LD scanner for sampling large numbers of sarcomeres in thick whole muscle longitudinal sections. Sections of high optical quality and fixation were produced from tibialis anterior and extensor digitorum longus muscles of Sprague-Dawley rats (N=6). Scans produced two-dimensional Ls maps, capturing >85% of the muscle area per section. Individual Ls measures generated by automatic LD and bright-field microscopy showed excellent agreement over a large Ls range (ICC>0.93). Two-dimensional maps also revealed prominent regional Ls variations across muscles.

Resonant reflection spectroscopy of biomolecular arrays in muscle.

Sarcomeres, the functional units of contraction in striated muscle, are composed of an array of interdigitating protein filaments. Direct interaction between overlapping filaments generates muscular force, which produces animal movement. When filament length is known, sarcomere length successfully predicts potential force, even in whole muscles that contain billions of sarcomere units. Inability to perform in vivo sarcomere measurements with submicrometer resolution is a long-standing challenge in the muscle physiology field and has hampered studies of normal muscle function, adaptation, injury, aging, and disease, particularly in humans. Here, we develop theory and demonstrate the feasibility of to our knowledge a new technique that measures sarcomere length with submicrometer resolution. In this believed novel approach, we examine sarcomere structure by measuring the multiple resonant reflections that are uniquely defined by Fourier decomposition of the sarcomere protein spatial framework. Using a new supercontinuum spectroscopic system, we show close agreement between sarcomere lengths measured by resonant reflection spectroscopy and laser diffraction in an ensemble of 10 distinct muscles.

Polarization gating enables sarcomere length measurements by laser diffraction in fibrotic muscle.

Raman spectroscopy (RS) has been extensively used to characterize bone composition. However, the link between bone biomechanics and RS measures is not well established. Here, we leveraged the sensitivity of RS polarization to organization, thereby assessing whether RS can explain differences in bone toughness in genetic mouse models for which traditional RS peak ratios are not informative. In the selected mutant mice­activating transcription factor 4 (ATF4) or matrix metalloproteinase 9 (MMP9) knock-outs­toughness is reduced but differences in bone strength do not exist between knock-out and corresponding wild-type controls. To incorporate differences in the RS of bone occurring at peak shoulders, a multivariate approach was used. Full spectrum principal components analysis of two paired, orthogonal bone orientations (relative to laser polarization) improved genotype classification and correlation to bone toughness when compared to traditional peak ratios. When applied to femurs from wild-type mice at 8 and 20 weeks of age, the principal components of orthogonal bone orientations improved age classification but not the explanation of the maturation-related increase in strength. Overall, increasing polarization information by collecting spectra from two bone orientations improves the ability of multivariate RS to explain variance in bone toughness, likely due to polarization sensitivity to organizational changes in both mineral and collagen.