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Functional imaging of muscle cells by second harmonic generation

The intrinsically ordered arrays of proteins (mainly actin and myosin) constituting the myofibrils within muscle cells are at the basis of a strong Second Harmonic Generation (SHG) from muscle fibers and isolated myofibrils. We have characterized the SHG signal with regard to its polarization and potential source within the muscle cell. The lateral resolution that can be achieved through SHG imaging of muscle strongly depends on sample depth. In fact, a comparison between intact muscle fibers and single myofibrils demonstrates that, whereas in both cases the alternation of dark I bands and bright A bands is visible, the contours of these bands are much better resolved in myofibrils than in fibers. Further, imaging of myofibrils revealed the presence of a darker zone in the centre of the A band. These effects of scattering by tissue on the image resolution were also studied with regard to the polarization of the SHG signal. The polarization-dependence of SHG intensity represents a powerful tool for the investigation of the structural dynamics occurring in the emitting proteins during the active cycle of muscle contraction. The prospective to perform functional studies requires a complete characterization of the effects of scattering and possibly multiple emitting populations on the measured SHG signal. Also, SHG is extremely sensitive to the degree of order present in the filament array, offering an interesting potential in the development of non-invasive tools for the diagnosis of degenerative diseases affecting skeletal muscles.