In imaging anisotropic samples with optical microscopy, a controlled, polarized light source can be used to gain molecular information of fibrous materials such as muscles and collagen fibers. However, the delivery of the polarized excitation light source in a system such as a laser scanning optical microscope often encounters the problem of the polarization ellipticity altering effects of the optical components. Using a half-wave plate and a quarter-wave plate, we demonstrate that the polarization ellipticity altering effect of the dichroic mirror in an epi-illuminated multiphoton laser scanning microscope can be corrected, and that this approach can be used to obtain polarized second-harmonic generation (SHG) images of rat tail tendon and mouse leg muscle. The excitation polarization dependence of the SHG intensity is fitted to determine the ratio of the second-order susceptibility tensor elements associated with type I collagen in the rat tail tendon and myofibril in the mouse leg muscle. Our methodology can be applied to polarized SHG imaging without sample rotation. This approach has great potential for imaging noncentrosymmetric biological samples, providing structural information on the molecular scale in addition to morphological information of tissues.