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

Abstract

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 ¼ 0.12 μm for single-band intensity profiles when fixing the A-band length (A) to A ¼ 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 ¼ n 2ω − n ω ) 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 ¼ 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 ¼ 1.40 AE 0.04 μm and M ¼ 0.07 AE 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.

Fully automated muscle quality assessment by Gabor filtering of second harmonic generation images

“…Sarcomere analysis based on manually selected profiles was done as previously described. 14 In short, 2728 intensity profiles were manually selected from 68 SHG images, taken from both healthy (n ¼ 5) and EAE (n ¼ 8) animals. Each profile was analyzed using the supramolecular model developed by Rouède et al, 8 yielding the sarcomere length L and the disorder-related M-band size M. Based on our previous observations, the analysis was done using an A-band length of A ¼ 1.40 μm, a refractive index (RI) at the illumination wavelength n ω ¼ 1.39, and an RI at the SHG wavelength n 2ω ¼ 1.41.…”

“…14 For the selected objective, the Gaussian point spread function (PSF) has a lateral size of w xy ¼ 0.59 μm and an axial size of w z ¼ 3.45 μm (half width at e −2 ). 14…”

“…12,13 The thick filament misalignment due to absence of M-proteins is known to lie at the basis of clear transition from normal single-band to double-band SHG intensity profiles. 8,14 Based on a supramolecular sarcomere model to calculate the expected SHG intensity profiles, the degree of thick filament misalignment can be quantified by means of an apparent increase in the centrosymmetric M-band size. 8,14 Currently, this degree of sarcomere disorder is mostly obtained by analyzing local, manually selected one-dimensional sarcomere intensity profiles.…”

“…8,14 Based on a supramolecular sarcomere model to calculate the expected SHG intensity profiles, the degree of thick filament misalignment can be quantified by means of an apparent increase in the centrosymmetric M-band size. 8,14 Currently, this degree of sarcomere disorder is mostly obtained by analyzing local, manually selected one-dimensional sarcomere intensity profiles. On the other hand, based on the distance-dependent correlation value of the gray-level co-occurrence matrix, Liu et al 15 suggested a method for assessing sarcomere regularity of an entire SHG image.…”

“…First, the data are used to indicate how the Gabor values should be interpreted based on representative SHG intensity profiles. Next, the relation between the disorder-related M-band size 8,14 and the normalized Gabor value is studied to justify the use of the Gabor approach for assessing sarcomere regularity. Finally, we study the correlation between the EAE severity and the sarcomere regularity quantified by our normalized Gabor value.…”