Minimally Invasive Principles and Technology to Measure Dynamic Skeletal Muscle Sarcomere Length
Skeletal muscle force production results from interaction between actin and myosin protein filaments in repeating units called sarcomeres. Sarcomere force is transmitted through the basement membrane via protein complexes to a network of connective tissue. Connective tissues transmit force to bone. Typically, muscles are characterized in vitro but this may be insufficient because stresses produced in isolated fibers or muscle bundles differ from whole muscle dynamics. Furthermore, comparisons between normal and diseased muscle states are impossible to make without a proper normalization factor such as sarcomere length. These limitations point to the need for obtaining data in vivo during dynamic movement in the intact muscle. Several tools are available to collect in vivo sarcomere length data, but all fall short of the necessary technical requirements for sarcomere dynamics. To address this need, this dissertation leverages optical communication technology to create new principles and tools to analyze skeletal muscle protein structure.
First, this dissertation improves the sensitivity of existing laser diffraction to measure sarcomere length in fibrotic muscle. Due to an increase in connective tissue, fibrotic muscle scatters laser light and buries the needed diffraction pattern that is used to calculate sarcomere length. Polarization gating is used to filter between scattered and diffracted light to recover the signal and enable sarcomere length measurements.
Second, this dissertation introduces a fundamentally new method to measure sarcomere structure termed resonant reflection spectroscopy (RRS). RRS addresses the challenge of in vivo sarcomere length data collection by illuminating sarcomeres and collecting signals through a single minimally invasive fiber optic probe. Relationships between signal and sarcomere structure are discussed theoretically and simulated in custom made computer code.
Third, proof of concept is demonstrated for RRS measurements of sarcomere length. A new system, based upon optical communication technology, is built and used to measure sarcomere length in an ensemble of muscles. Validation is demonstrated by comparing sarcomere lengths measured by RRS and traditional laser diffraction. Very high agreement between the methods was achieved.
Fourth, proof of feasibility is demonstrated for minimally-invasive profiling of sarcomere length in muscle during activity and movement. The proof of feasibility system combined RRS with optical frequency domain interferometry (OFDI) to enable profiling sarcomere lengths across millimeters of tissue. Sarcomere lengths are measured with nanometer resolution during passive strain, twitch and tetanic contractions.
Lastly, RRS requires bandwidths and speeds that are inaccessible from commercial laser emitters, and so design rules are developed for swept-pump fiber optic parametric oscillator (FOPO) systems. A FOPO system is demonstrated with 422 nm bandwidth and 14 kHz sweep speed.
Taken together, these demonstrations establish a fundamentally new ability to study movement disorders, including patients with cerebral palsy, stroke, Parkinson’s disease and muscular dystrophy.