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, noninva- siveness, high specificity, and high penetrability. For a bio- medical application, the epicollection of backward propa- gating 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 ra- diation patterns of myosin-based muscle fibers and col- lagen 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 prefer- entially 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. © 2009 Society of Photo-Optical Instrumentation Engi-