Abstract
Polarimetric second harmonic generation (SHG) microscopy is a label-free ultrastructural imaging technique that is applied to study molecular organization in striated muscle structure. Wild type OreR and UNC45 knockdown Drosophila melanogaster somatic larva body wall muscles are used to elucidate the influence of myosin organization on SHG imaging response. Structural changes due to varying myosin content are studied with linear polarization-in polarization-out (PIPO) SHG microscopy. PIPO SHG microscopy revealed changes in the striated pattern of somatic muscles and a reduced signal intensity correlated with reduced order of myosin filaments. The achiral and chiral susceptibility component ratios R- and C-ratios, respectively, were deduced. R-ratio values were higher in the UNC45 knockdown larva mutant compared to control, suggesting that in addition to myosin, another protein structure provide basis for a second harmonic response. The SHG active protein structure is located in the region of isotropic bands of the striated muscles. The measured C-ratio from the muscle fibers appeared to be small. Nonetheless, the wide distribution of C-ratio values of myosin filaments could be observed in the wild-type OreR muscles. Overall, this technique provides insight on how second harmonic properties change with myosin filament structure.
Highlights
second harmonic generation (SHG) is a label-free image contrast mechanism for visualizing non-centrosymmetric biological structures with nonlinear optical microscopy [1]
Quantitative polarization-based SHG microscopy was used to characterize the changes in UNC45 knockdown muscles
R-ratio was higher in UNC45 knockdown muscles that were severely affected, whereas muscles that still exhibited a striated pattern R-ratio was found to be lower in A-bands and not significantly different from OreR muscles
Summary
SHG is a label-free image contrast mechanism for visualizing non-centrosymmetric biological structures with nonlinear optical microscopy [1]. SHG microscopy enables non-invasive in vivo visualization of striated muscles due to myosin’s helical non-centrosymmetric structure and its crystalline arrangement [5,6,7,8]. The main contractile unit in striated muscle is the sarcomere, which has a crystalline arrangement of protein filaments that are Polarimetric SHG Imaging of Muscle indispensable for contraction. The A bands are defined as the regions spanning the entire length of the thick filaments, or myosin filaments. These bipolar filaments are formed from the bundling of myosin II molecules, which are characterized by two distinct globular heads that interact with actin during contraction [11]
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