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We show that the canonical single frequency sarcomeric SHG intensity pattern (SHG-IP) of control muscles is converted to double frequency sarcomeric SHG-IP in preserved mdx mouse gastrocnemius muscles in the vicinity of necrotic fibers. These double frequency sarcomeric SHG-IPs are often spatially correlated to double frequency sarcomeric two-photon excitation fluorescence (TPEF) emitted from Z-line and I-bands and to one centered spot SHG angular intensity pattern (SHG-AIP) suggesting that these patterns are signature of myofibrillar misalignement. This latter is confirmed with transmission electron microscopy (TEM). Moreover, a good spatial correlation between SHG signature of myofibrillar misalignment and triad reduction is established. Theoretical simulation of sarcomeric SHG-IP is used to demonstrate the correlation between change of SHG-IP and -AIP and myofibrillar misalignment. The extreme sensitivity of SHG microscopy to reveal the submicrometric organization of A-band thick filaments is highlighted. This report is a first step toward future studies aimed at establishing live SHG signature of myofibrillar misalignment involving excitation contraction defects due to muscle damage and disease.

SHG angular intensity pattern (SHG-AIP) of healthy and proteolysed muscle tissues are simulated and imaged here for the first time to our knowledge. The role of the spatial distribution of second-order nonlinear emitters on SHG-AIP is highlighted. SHG-AIP with two symmetrical spots is found to be a signature of healthy muscle whereas SHG-AIP with one centered spot in pathological mdx muscle is found to be a signature of myofibrillar disorder. We also show that SHG-AIP provides information on the three-dimensional structural organization of myofibrils in physiological and proteolysed muscle. Our results open an avenue for future studies aimed at unraveling more complex physiological and pathological fibrillar tissues organization.

A theoretical far-field second harmonic generation (SHG) imaging radiation pattern is calculated for muscular myosin taking into account both Gouy effect and light diffraction under high focusing excitation. Theoretical analysis, in agreement with experimental results obtained on healthy Xenopusmuscles, shows that the increase on intensity at the middle of the sarcomeric SHG intensity pattern is generated by an off-axis constructive interference related to the specific antipolar distribution of myosin molecules within the sarcomere. The best fit of the experimental sarcomeric SHG intensity pattern was obtained with an estimated size of antiparallel, intrathick filaments' packing-width of 115 ± 25 nm localized at the M-band. During proteolysis, experimental sarcomeric SHG intensity pattern exhibits decrease on intensity at the center of the sarcomere. An effective intra- and interthick filaments centrosymmetry of 320 ± 25 nm, in agreement with ultrastructural disorganization observed at the electron microscopy level, was necessary to fit the experimental sarcomeric SHG intensity pattern. Our results show that sarcomeric SHG intensity pattern is very sensitive to misalignment of thick filaments and highlights the potential usefulness of SHG microscopy to diagnose proteolysis-induced muscular disorders.

Femtosecond laser at 780 nm excitation wavelength was used to photo-convert the physiological sarcomeric single band (SB) second harmonic generation (SHG) pattern into double band (DB) in Xenopus laevis premetamorphic tail muscles. This photo-conversion was found to be a third order non-linear optical process and was drastically reduced at 940 nm excitation wavelength. This effect was no longer observed in paraformaldehyde fixed muscles and was enhanced by hydrogen peroxide. The action of hydrogen peroxide suggests that reactive oxygen species (ROS) could contribute to this photo-conversion. These results demonstrate that sarcomeric DB SHG pattern is a marker of sarcomere photodamage in xenopus tadpole muscles and highlight the need of being very careful at using two-photon excitation while observing living tissues. Moreover they open new avenues for in situ intravital investigation of oxidative stress effects in muscle dysfunctions and diseases.