Research Papers: Imaging

On the interpretation of second harmonic generation intensity profiles of striated muscle

[+] Author Affiliations
Rik Paesen, Sophie Smolders, Kristof Notelaers, José Manolo de Hoyos Vega, Virginie Bito, Marcel Ameloot

Hasselt University, Biomedical Research Institute, Agoralaan Building C, Diepenbeek 3590, Belgium

Inez Wens, Bert O. Eijnde, Dominique Hansen

Hasselt University, Biomedical Research Institute, Agoralaan Building C, Diepenbeek 3590, Belgium

Hasselt University, REVAL—Rehabilitation Research Center, Agoralaan Building A, Diepenbeek 3590, Belgium

J. Biomed. Opt. 20(8), 086010 (Aug 17, 2015). doi:10.1117/1.JBO.20.8.086010
History: Received April 20, 2015; Accepted July 21, 2015
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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=0.12μm for single-band intensity profiles when fixing the A-band length (A) to A=1.6μm, 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±0.04μm and M=0.07±0.05μm 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.

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© 2015 Society of Photo-Optical Instrumentation Engineers

Citation

Rik Paesen ; Sophie Smolders ; Inez Wens ; Kristof Notelaers ; José Manolo de Hoyos Vega, et al.
"On the interpretation of second harmonic generation intensity profiles of striated muscle", J. Biomed. Opt. 20(8), 086010 (Aug 17, 2015). ; http://dx.doi.org/10.1117/1.JBO.20.8.086010


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