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Journal of Biomedical Optics

On the interpretation of second harmonic generation intensity profiles of striated muscle
Author(s): Rik Paesen; Sophie Smolders; Inez Wens; Kristof Notelaers; José Manolo de Hoyos Vega; Virginie Bito; Bert O. Eijnde; Dominique Hansen; Marcel Ameloot
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Paper Abstract

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.

Paper Details

Date Published: 17 August 2015
PDF: 8 pages
J. Biomed. Opt. 20(8) 086010 doi: 10.1117/1.JBO.20.8.086010
Published in: Journal of Biomedical Optics Volume 20, Issue 8
Show Author Affiliations
Rik Paesen, Univ. Hasselt (Belgium)
Sophie Smolders, Univ. Hasselt (Belgium)
Inez Wens, Hasselt University (Belgium)
Kristof Notelaers, Hasselt University (Belgium)
José Manolo de Hoyos Vega, Hasselt Univ. (Belgium)
Virginie Bito, Univ. Hasselt (Belgium)
Bert O. Eijnde, Univ. Hasselt (Belgium)
Dominique Hansen, Univ. Hasselt (Belgium)
Marcel Ameloot, Univ. Hasselt (Belgium)

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