Dynamic investigation of Drosophila myocytes with second harmonic generation microscopy

Abstract

The functional dynamics and structure of both larval and adult <i>Drosophila melanogaster</i> muscle were investigated with a nonlinear multimodal microscope. Imaging was carried out using a home built microscope capable of recording the multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation signals simultaneously at a scanning rate of up to ~12 frames/sec. The sample was excited by a home built femtosecond Ti:Sapphire laser at 840 nm, or by a Yb-ion doped potassium gadolinium tungstate (Yb:KGW) crystal based oscillator at 1042 nm. There was no observable damage detected in the myocyte after prolonged scanning with either of the lasers. Microscopic second harmonic generation (SHG) appears particularly strong in the myocytes. This allows the fast contraction dynamics of the myocytes to be followed. The larger sarcomere size observed in the larvae myocytes is especially well suited for studying the contraction dynamics. Microscopic imaging of muscle contractions showed different relaxation and contraction rates. The SHG intensities were significantly higher in the relaxed state of the myocyte compared to the contracted state. The imaging also revealed disappearance of SHG signal in highly stretched sarcomeres, indicating that SHG diminishes in the disordered structures. The study illustrates that SHG microscopy, combined with other nonlinear contrast mechanisms, can help to elucidate physiological mechanisms of contraction. This study also provides further insight into the mechanisms of harmonic generation in biological tissue and shows that crystalline arrangement of macromolecules has a determining factor for the high efficiency second harmonic generation from the bulk structures.