17 August 2015 On the interpretation of second harmonic generation intensity profiles of striated muscle
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Recently, a supramolecular model was developed for predicting striated skeletal muscle intensity profiles obtained by label-free second harmonic generation (SHG) microscopy. This model allows for a quantitative determination of the length of the thick filament antiparallel range or M-band (M), and results in M=0.12  μm for single-band intensity profiles when fixing the A-band length (A) to A=1.6  μm, a value originating from electron microscopy (EM) observations. Using simulations and experimental data sets, we showed that the objective numerical aperture (NA) and the refractive index (RI) mismatch (Δn=n2ωnω) between the illumination wave (ω) and the second harmonic wave (2ω) severely affect the simulated sarcomere intensity profiles. Therefore, our recovered filament lengths did not match with those observed by EM. For an RI mismatch of Δn=0.02 and a moderate illumination NA of 0.8, analysis of single-band SHG intensity profiles with freely adjustable A- and M-band sizes yielded A=1.40±0.04  μm and M=0.07±0.05  μm for skeletal muscle. These lower than expected values were rationalized in terms of the myosin density distribution along the myosin thick filament axis. Our data provided new and practical insights for the application of the supramolecular model to study SHG intensity profiles in striated muscle.
Rik Paesen, Rik Paesen, Sophie Smolders, Sophie Smolders, Inez Wens, Inez Wens, Kristof Notelaers, Kristof Notelaers, José Manolo de Hoyos Vega, José Manolo de Hoyos Vega, Virginie Bito, Virginie Bito, Bert O. Eijnde, Bert O. Eijnde, Dominique Hansen, Dominique Hansen, Marcel Ameloot, Marcel Ameloot, } "On the interpretation of second harmonic generation intensity profiles of striated muscle," Journal of Biomedical Optics 20(8), 086010 (17 August 2015). https://doi.org/10.1117/1.JBO.20.8.086010 . Submission:

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