Ultrastructural characterization of individual myofibrils using second harmonic generation

Abstract

Second harmonic generation (SHG) is a non-linear optical effect in which two identical incident photons interact with a material to generate one photon at twice the frequency of one incident photon. Polarization-resolved second harmonic generation microscopy (PSHG) has been used in biomedical research to study collagen, muscle, and a variety of other biological tissues. In this work individual myofibrils were imaged with PSHG microscopy, and the optical parameter r, which is related to molecular disorder, is reported. The relationship between contractile state of individual myofibrils and r was also investigated. The results of this study show that individual myofibrils dissected from Drosophila melanogaster can be successfully imaged using PSHG. Individual myofibrils dissected and fixed at physiological ATP concentration and imaged in water were found to have a mean ρ value of 0.544 ± 0.03. A gradient in ρ value perpendicular to the direction of the sarcomere was observed in fibrils in this population. In the A-band, I-band, and M-line of the sarcomere the average ρ values are reported in both the relaxed and rigor state. The mean ρ value of the A-band was found to be significantly higher than the M-line and I-band in the relaxed state. In the contracted state the level of disorder in the three regions was not significantly different. The level of disorder increased in all three regions as ATP concentration decreased.