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

INTRODUCTION

Second harmonic generation (SHG) imaging, a recently introduced nonlinear optical microscopy technique, is based on interaction of a strong laser beam with highly polarizable matter in a noncentrosymmetric molecular organization. Such interaction results in emission of photons with exactly twice the energy of the incident laser (see reviews (1,2)). Many animal tissue structures can be imaged via SHG microscopy, by virtue of intrinsic contrast generated by filamentous proteins: collagen fibrils in connective tissues, microtubules, and the actomyosin lattice of muscle cells (3-16). SHG microscopy benefits from intrinsic optical sectioning, deep penetration into three-dimensional samples, and the presence of endogenous sources in live, untreated specimens. SHG can be imaged simultaneously with distinct two-photon-excited fluorescence (2PEF) signals from one or more endogenous or exogenous labels (17). These attributes underlie the potential of SHG imaging in biomedical applications including native pathology and high-resolution in vivo imaging or spectroscopy via fiber-based laser-scanning probes (18).

Knowing the source of the SHG will be a fundamental requirement for any such applications. Whereas the component proteins that give rise to SHG in collagen fibrils and microtubules are clearly collagen and tubulin, the muscle sarcomere has a more complex structure comprising three distinct forms of major filaments and dozens of proteins (19). Thick myosin filaments, thin actin filaments, titin filaments, or the combination of these, could comprise the SHG harmonophore (Fig. 1). Because these filaments undergo changes in conformation and the extent of their interaction during the contraction and extension of muscle, it is possible that SHG might vary quantifiably during contraction, allowing the use of SHG spectroscopy to measure sarcomeric activity. In addition, polarization or intensity of the SHG signal may quantifiably indicate the composition of new or changing myofibrils.

Previous characterization of the birefringence of myofibrils has concluded that contrast within the anisotropic A band, which corresponds spatially to sarcomeric SHG, is influenced by the overlap of thick and thin filaments (20,21). The similarities between SHG and polarization microscopy images are suggestive of similar sources of contrast. Yet, the two modalities have already been shown to highlight non-identical sets of structures in nematode muscle cells (5). Correlations between these two optical phenomena without direct experimentation is therefore not necessarily tenable.

Previous evidence from our group has indicated that genetic disruption of myosin heavy chain genes (MHC) in Caenorhabditis elegans reduces sarcomeric SHG and that the localization of SHG in C. elegans muscle corresponded to a region containing thick filaments (5). However, the striation pattern of these mutant animals developed completely abnormally, preventing us from concluding absolutely that MHC alone, and not other features of normal sarcomeric structure, was the critical harmonophore for SHG.

Because of the crucial importance of understanding the molecular source of sarcomeric SHG for any interpretation of experimental or medical imaging data, we have undertaken here an exhaustive characterization of SHG from isolated myofibrils. These preparations have allowed us to perform biochemical and pharmacological manipulations of the various sarcomeric filaments and myosin motor domains while imaging, and enabled high-resolution analysis of the polarization anisotropy of SHG within individual sarcomeres. This work leads to our conclusion that SHG arises from within the coiled rod region of myosin thick filaments and that SHG, although indicative of contraction and extension in images of muscle, does not depend upon the functional state of myosin head domains or the actin filaments with which they interact.

MATERIALS AND METHODS

Experimental microscope setup and imaging conditions