Spatial orientation mapping of fibers using polarization‐sensitive second harmonic generation microscopy

Abstract

In this work, we present a non-invasive approach to determine azimuth and elevation angles of collagen fibers capable of generating second harmonic signal. The azimuth angle was determined using the minimum of second harmonic generation (SHG) signal while rotating the plane of polarization of excitation light. The elevation angle was estimated from the ratio of the minimal SHG intensity to the intensity when laser polarization and fiber directions were parallel to each other using experimentally determined calibration curve. Pixel-resolution images of collagen fiber spatial orientation in tendon from bovine leg, chicken leg, and chicken skin were acquired using our approach of SHG polarization-resolved microscopy.

Three-Dimensional Geometry of Collagenous Tissues by Second Harmonic Polarimetry

“…Generally multiple scans are performed to determine the parallel and perpendicular components of the SHG signal; Erikson’s equations,or variations of them, are then used to calculate the out-of-plane orientation angle on a per pixel basis. This approach has been used to visualize spatial relationships in a variety of tissues, including tendon, skin and cornea 26–28 .…”

Imaging of biological tissues with pixel-level analysis of second-order susceptibility

“…84 Moreover, developmental efforts of 3D analysis have been carried out for biological structures, such as inherent crystallinity of amylopectin in starch grain, and orientation mapping for collagen fibril in mammalian tissues such as bovine legs, chicken legs, and chicken skin by extracting information from the elevation angle. 85,86 For thin tissue specimens, BSHG is attributed to the backscattering of FSHG, whereas in thick tissue, BSHG is less than 1% of FSHG, which yields more informative images than BSHG. 87,88 Based on the difference in coherence interaction lengths for FSHG and BSHG, selective SHG imaging has been demonstrated to discriminate collagen fibrils from muscle fibers by BSHG and FSHG imaging, respectively, which suggests the usefulness of backward and forward imaging for different applications.…”

Accumulating advantages, reducing limitations: Multimodal nonlinear imaging in biomedical sciences – The synergy of multiple contrast mechanisms

“…Polarization sensitive SHG enables visualization of the fiber direction. Maximal SHG signal is emitted, when the polarization direction is orthogonal to the fiber axis, which allows for direction mapping of the fiber orientation in tendon as shown in Figure 2C [ 35], in the cornea [36], but also for determining the ordering of cholesterol crystals [37]. Similarly, also other techniques like SRS are sensitive to the input polarization, which provides additional information on the molecular ordering, e.g., of cholesterol crystals [37].…”

“…Maximal SHG signal is emitted, when the polarization direction is orthogonal to the fiber axis. left, circularly polarized light SHG of bovine tendon at the surface, mid: azimutal angle map, right: two-colour SHG image at two depths 1,5 mm apart [35].…”

“…The same is true for visualizing the distinct morphology and characteristic structures of the inner organs and disease related modifications, e.g., for kidney tumors [114], pancreas and pancreatic cancer [132,133], bladder [134], ovary and ovarian tumors [135][136][137], and lung carcinoma [138]. In addition the structure of the musculoskeletal system has been visualized, in particular the structure of muscle [109] and tendon [34,35], but also of bones and cartilage [59,139]. Highly important is visualization of the central nervous system due to its importance for controlling the living pro- Figure 6 Comparison of staining histopathology with multimodal nonlinear imaging.…”