Characterization of the Myosin-Based Source for Second-Harmonic Generation from Muscle Sarcomeres

Abstract

Several biologically important protein structures give rise to strong second-harmonic generation (SHG) in their native context. In addition to high-contrast optical sections of cells and tissues, SHG imaging can provide detailed structural information based on the physical constraints of the optical effect. In this study we characterize, by biochemical and optical analysis, the critical structures underlying SHG from the complex muscle sarcomere. SHG emission arises from domains of the sarcomere containing thick filaments, even within nascent sarcomeres of differentiating myocytes. SHG from isolated myofibrils is abolished by extraction of myosin, but is unaffected by removal or addition of actin filaments. Furthermore, the polarization dependence of sarcomeric SHG is not affected by either the proportion of myosin head domains or the orientation of myosin heads. By fitting SHG polarization anisotropy readings to theoretical response curves, we find an orientation for the elemental harmonophore that corresponds well to the pitch of the myosin rod alpha-helix along the thick filament axis. Taken together, these data indicate that myosin rod domains are the key structures giving SHG from striated muscle. This study should guide the interpretation of SHG contrast in images of cardiac and skeletal muscle tissue for a variety of biomedical applications.

In vivo imaging of skeletal muscle in mice highlights muscle defects in a model of myotubular myopathy

“…Second harmonic generation was detected with a trans-detector through a single-band bandpass filter 470/22 nm. As described previously, second harmonic generation occurs mostly from 2PEM of non-centrosymetric structures, and is restricted to collagen fibers and striated muscle myosin rod domains 44 in mammalian soft tissues. Consecutive images of 512 £ 512 pixels (pixel size: 0.3 mm in x/y, 1 mm in z) were acquired at 0.4 frames/s.…”

“…SHG does not require exogenous labeling as it is based on the fluorescence of non-centrosymmetric structures as myosin rod domains that are parallelly aligned in the sarcomere. 32,44,46 Chemical dyes have the advantage to cover a very large spectrum of structures and organelles, are easy to use, and are partially applied in clinical practice. The fluorescence-coupled molecules offer the possibility to tag and follow nearly any desirable molecule, and recombinant DNA constructs open the perspective to utilize advanced imaging modes as photoactivation and optogenetics.…”

Invertebrate muscles: Thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

“…Second-harmonic generation studies in mollusc show that the myosin tails continue to be in an α-helix when on the filament surface (Plotnikov et al, 2006), but this technique provides no information about their arrangement on it. Work in Pecten and Crassostrea indicates a helical arrangement of the heads, but does not provide information about how many helices are present or their spacing (Elliott, 1971(Elliott, , 1974.…”

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy

“…Another equally powerful NLO imaging technique providing morpho-functional information on biological tissues is represented by second-harmonic generation (SHG) microscopy [3,51]. In fact, SHG microscopy can provide background free morphological and functional images by revealing the distribution of anisotropic biological structures with large hyperpolarizability [52,53], such as collagen [54,55], muscle [56][57][58][59][60], or microtubules [61]. SHG is a nonlinear second order optical process occurring in noncentrosymmetric systems with a large hyperpolarizability.…”