Selective imaging in second-harmonic-generation microscopy with anisotropic radiation

As a novel modality of optical microscopy, second-harmonic generation (SHG) provides attractive features including intrinsic optical sectioning, noninvasiveness, high specificity, and high penetrability. For a biomedical application, the epicollection of backward propagating SHG is necessary. But due to phase-matching constraint, SHG from thick tissues is preferentially forward propagation. Myosin and collagen are two of the most abundant fibrous proteins in vertebrates, and both exhibit a strong second-harmonic response. We find that the radiation patterns of myosin-based muscle fibers and collagen fibrils are distinct due to coherence effects. Based on these asymmetric radiation patterns, we demonstrate selective imaging between intertwining muscle fibers and type I collagen fibrils with forward and backward SHG modalities, respectively. Thick muscle fibers dominate the forward signal, while collagen fibril distribution is preferentially resolved in the backward channel without strong interference from muscle. Moreover, we find that well-formed collagen fibrils are highlighted by forward SHG, while loosely arranged collagen matrix is outlined by backward signal.