Non-invasive label-free assessment of contraction induced damage to skeletal muscle structure using second harmonic generation (SHG) microscopy

Second harmonic generation (SHG) microscopy is a second-order nonlinear optical scattering process that can nondestructively image myosin filament microarchitecture up to 500 μm in depth due to its high spatial resolution, optical sectioning capabilities, and elastic scattering properties. The distribution, alignment, and spatial orientation of myosin filaments largely dictates the mechanical properties of skeletal muscle. Noninvasive quantification of these metrics in-vivo or in-vitro could be indicative of skeletal muscle function and viability. Here, we present a novel algorithm to quantitate sarcomere length with high spatial resolution and accuracy when compared to traditional approaches. Our approach runs in real-time and is less sensitive to instrumental noise and artifacts that shade regions of the image. We have tested this technique on ex-vivo SHG images of rat lumbrical muscles that have been damaged following standardized lengthening contraction protocols. Our approach shows accurate quantitation of sarcomere lengths within 1% compared to manual calculations. This algorithm can be used to reliably analyze SHG images in realtime to assess the structure and function of skeletal muscle tissue in a non-invasive, quantitative, and sterile manner.