Abstract
Optical second- and third-harmonic generations have attracted a lot of attention in the biomedical imaging research field recently due to their intrinsic sectioning ability and noninvasiveness. Combined with near-infrared excitation sources, their deep-penetration ability makes these imaging modalities suitable for tissue characterization. In this article, we demonstrate a polarization harmonics optical microscopy, or P-HOM, to study the nonlinear optical anisotropy of the nanometer-scaled myosin and actin filaments inside myofibrils. By using tight focusing we can avoid the phase-matching condition due to micron-scaled, high-order structures in skeletal muscle fibers, and obtain the submicron-scaled polarization dependencies of second/third-harmonic generation intensities on the inclination angle between the long axes of the filaments and the polarization direction of the linear polarized fundamental excitation laser light. From these dependencies, detailed information on the tensor elements of the second/third-order nonlinear susceptibilities contributed from the myosin/actin filaments inside myofibrils can thus be analyzed and obtained, reflecting the detailed arrangements and structures of the constructing biomolecules. By acquiring a whole, nonlinearly sectioned image with a submicron spatial resolution, we can also compare the polarization dependency and calculate the nonlinear susceptibilities over a large area of the tissue at the same time-which not only provides statistical information but will be especially useful with complex specimen geometry.