Question: 

Second harmonic generation (SHG) is a nonlinear polarization effect that can be observed in several biological structures like filaments of collagen-I or skeletal and cardiac muscle myosin-II upon excitation with a pulsed infrared laser. SHG microscopy can provide high contrast images of striated muscle and connective tissue without the need for extrinsic staining. As SHG is a coherent optical process occurring directly in the protein structures, the signals do not only contain information about the location of myosin molecules, but also on their orientation and on local symmetry conditions. We were interested in whether SHG microscopy could be used to distinguish different cross-bridge states in isolated myofibrils.

Methodology: 

We measured SHG signal intensities from myofibrils in either the ATP-free rigor state or a relaxed state with ATP present and recorded characteristic signal dependencies on the polarization angle of the excitation laser beam relative to the filament axis. By means of mathematical modeling, we identified these polarization dependencies with a single parameter g that is directly linked to the physical property of the second-order susceptibility c⁽²⁾ of the myosin filaments.

Results: 

Polarization dependencies differed significantly for myofibrils in the relaxed and the rigor state and resulted in two distinct specific values for g. Mathematical modeling further provides indications that this difference is due to a bending of the myosin S2 domain between the two states.

Conclusion: 

The presented method of polarization dependent SHG microscopy and the results found allow us to distinguish between different actin-bound states of native myosin ensembles and to study molecular kinetics on a two-photon microscope.