Three distinct sarcomeric patterns of skeletal muscle revealed by SHG and TPEF Microscopy

Abstract

Abstract:We have extensively characterized the sarcomeric SHG signal as a function of animal species (rat versus xenopus), age (adult versus larval) and tissue preparation (fixed or fresh) and we found that the main feature of this signal is a single peak per mature sarcomere (about 85% of all sarcomeres). The remaining (15%) was found to be either double peak per mature sarcomere or mini sarcomeres (half of a sarcomere) using α-actinin immuno detection of the Z-band. The mini sarcomeres are often found in region of pitchfork-like SHG pattern. We suggest that double peak SHG pattern could indicate regions of sarcomeric proteolysis whereas pitchfork-like SHG pattern could reveal sarcomeric assembly.

Myofibrillogenesis in live neonatal cardiomyocytes observed with hybrid two-photon excitation fluorescence-second harmonic generation microscopy

“…The image virtual stack was then processed by IMAGEJ software (http://rsbweb.nih.gov/ij/). Although no energy is absorbed during generation of second harmonics, 18 the incident laser may damage cardiomyocytes.…”

Imaging the bipolarity of myosin filaments with Interferometric Second Harmonic Generation microscopy

“…ISHG microscopy could be a useful technique to investigate the presence of a dip in the SHG signal of some sarcomere structures imaged with standard SHG microscopy [6][7][8][9][10]. These results highlight the bipolarity of myosin filaments in muscle tissue and show that muscle has a structural organization similar to a periodically poled crystal.…”

“…Since the myosin filaments have a noncentrosymmetric structural organization, muscle tissues can be imaged using Second Harmonic Generation (SHG) microscopy [6][7][8][9][10][11][12][13][14][15][16]. Other biological structures possess a noncentrosymmetric organization such as tissues rich in collagen type I/III proteins [14][15][16][17][18][19][20][21][22], collagen type 2 [22][23][24] and the microtubules within cells [25].…”

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

“…The well-defined properties of the SHG signal makes it convenient to study well-ordered structures, such as collagen type I rich structures, 1-4 microtubules inside mitotic spindles or neurons [5][6][7] and striated muscle containing myosin thick filaments. [8][9][10][11] SHG microscopy relies on the presence of noncentrosymmetric features, achieved through a high degree of order of polar biomolecules. This implies that even the smallest changes in this structural regularity have detrimental effects on the resulting SHG signals, making SHG microscopy one of the tools for early detection of the reorganization of SHG active molecules.…”

“…[9][10][11]17 Two SHG-based approaches have been used to quantify these striations. 9,16,18 Both methods are based on analyzing a one-dimensional intensity profile, mostly extracted from two-dimensional SHG images.…”