Intermyofilament dynamics of myocytes revealed by second harmonic generation microscopy.

Drosophila melanogaster larva myocytes are imaged with second harmonic generation (SHG) microscopy undergoing forced stretching and rhythmic contractions to determine the nature of the SHG signal. During stretching, double peaked SHG profiles of the anisotropic (A-) bands evolve into single peaks with a higher SHG intensity. The dip in the intensity profile at the center of the A-band is attributed to destructive interference from out-of-phase second harmonic radiating myosin molecules that, in the central region of myofilaments, are arranged antiparallel. An intensity increase at the center of the A-band appears during forced stretching due to a small, less than 100 nm, intermyofilament separation of the antiparallel myosin molecules leading to constructive interference of the SHG radiation. In addition, the same phenomenon occurs during periodic contractions of the myocyte, where an SHG intensity increase with the lengthening of sarcomeres is observed. The SHG intensity dependence on sarcomere length can be used for imaging myocyte contractions with low resolution microscopy, and can be applied for the development of diagnostic tools where monitoring of muscle contraction dynamics is required.