Structural origin of the drastic modification of second harmonic generation intensity pattern occurring in tail muscles of climax stages xenopus tadpoles

Abstract

Second harmonic generation (SHG) microscopy is a powerful tool for studying submicron architecture of muscles tissues. Using this technique, we show that the canonical single frequency sarcomeric SHG intensity pattern (SHG-IP) of premetamorphic xenopus tadpole tail muscles is converted to double frequency (2f) sarcomeric SHG-IP in metamorphic climax stages due to massive physiological muscle proteolysis. This conversion was found to rise from 7% in premetamorphic muscles to about 97% in fragmented muscular apoptotic bodies. Moreover a 66% conversion was also found in non-fragmented metamorphic tail muscles. Also, a strong correlation between predominant 2f sarcomeric SHG-IPs and myofibrillar misalignment is established with electron microscopy. Experimental and theoretical results demonstrate the higher sensitivity and the supra resolution power of SHG microscopy over TPEF to reveal 3D myofibrillar misalignment. From this study, we suggest that 2f sarcomeric SHG-IP could be used as signature of triad defect and disruption of excitation-contraction coupling. As the mechanism of muscle proteolysis is similar to that found in mdx mouse muscles, we further suggest that xenopus tadpole tail resorption at climax stages could be used as an alternative or complementary model of Duchene muscular dystrophy.

Functional second harmonic generation microscopy probes molecular dynamics with high temporal resolution

“…Since the discovery of second harmonic generation (SHG) signals generated in endogenous proteins like myosin in muscle [1][2][3][4][5][6] or collagen [2,7] various advances in this field of microscopy have been established: The intrinsic nature of the SHG signal allows for label free and high contrast imaging under in vivo [8] or ex vivo [2] conditions, thus making SHG microscopy a powerful tool in biomedical research. With muscle samples, structural SHG (sSHG) microscopy is used to investigate structural changes down to the sarcomeric level for a wide range of biomedical questions and applications [9][10][11][12][13]. Back scattered SHG signal [14,15] is of high interest for diagnostic applications and led to minimally invasive approaches [8].…”

Multiview deconvolution approximation multiphoton microscopy of tissues and zebrafish larvae

“…In the SV, constructive interference between emitters can enhance the signal between fibrils making their visual separation impossible. An example of such interference phenomena is the Vernier like artifacts reported for muscle fibres 34 , 35 and the elongated collagen fibrils seen in the Z-axis (Figs. 5 , 6 ).…”

Multiview Deconvolution Two-photon Laser Scanning Microscopy

“…In the SV, constructive interference between emitters can enhance the signal between brils making their visual separation impossible. An example of such interference phenomena is the Vernier like artifacts reported for muscle bres 29,30 and the elongated collagen brils seen in the Z-axis (Figs. 5, 6).…”