Quantitative analysis of collagen fiber organization in injured tendons using Fourier transform-second harmonic generation imaging

Abstract

Fourier transform-second harmonic generation (FT-SHG) imaging is used as a technique for evaluating collagenase-induced injury in horse tendons. The differences in collagen fiber organization between normal and injured tendon are quantified. Results indicate that the organization of collagen fibers is regularly oriented in normal tendons and randomly organized in injured tendons. This is further supported through the use of additional metrics, in particular, the number of dark (no/minimal signal) and isotropic (no preferred fiber orientation) regions in the images, and the ratio of forward-to-backward second-harmonic intensity. FT-SHG microscopy is also compared with the conventional polarized light microscopy and is shown to be more sensitive to assessing injured tendons than the latter. Moreover, sample preparation artifacts that affect the quantitative evaluation of collagen fiber organization can be circumvented by using FT-SHG microscopy. The technique has potential as an assessment tool for evaluating the impact of various injuries that affect collagen fiber organization.

Imaging the Nonlinear Susceptibility Tensor of Collagen by Nonlinear Optical Stokes Ellipsometry

“…Most commonly, the individual collagen fibers are considered to be lying within the image plane, resulting in polar tilt angles, q, of 90 , defined relative to the optical axis. This assumption holds reasonably well for highly aligned collagen tissues such as tendon samples sectioned along the major axis, and is used in numerous previous studies (15,29,30). However, for the meshlike collagen structures such as those found in skin tissues and basement membrane, the collagen fibers can no longer be considered to be lying within the imaging plane at each pixel.…”

Quantitative Analysis of Tendon Structural Integrity in Chickens using Fourier Transform-Second Harmonic Generation Imaging

“…By law, Novus International, Inc. is not required to maintain an Animal Care and Use Committee. However, the procedures followed to isolate the tendons for the current trial are the same as in our previous work with horse tendons [5] which was approved by the University of Illinois Animal Care and Use Committee.…”

“…Various techniques have been developed to assess collagen structural health. For example, researchers have used quantitative polarized-light microscopy to detect the degree of collagen parallelism in cartilage of porcine joints [4] and to evaluate collagen fiber organization in horse tendons [5]. Second harmonic generation using two-photon microscopy is the most recent modality in microscopy for analyzing collagen organization due to the strong non-centro-symmetric nature of the collagen's molecular arrangement in nature [6,7].…”

“…Second harmonic generation using two-photon microscopy is the most recent modality in microscopy for analyzing collagen organization due to the strong non-centro-symmetric nature of the collagen's molecular arrangement in nature [6,7]. Recently, we have developed a quantitative SHG imaging and analysis technique using Fast Fourier Transformation (FT-SHG) [8] to quantify and analyze collagen fiber organization in horse tendons [5,9]. Similar investigation was performed at different levels in a multitude of organisms where collagen plays a pivotal role in maintaining the integrity of tissue structure such as rat tail tendon [10] and porcine tendon [11].…”

“…Automated SHG imaging analysis has been developed to quantify the amount of matrix disorganization in rat patellar [12], rat tail tendons, mouse skin, bovine corneas, and human corneas [10]. FT-SHG can quantify the extent of collagen disorganization in different tissue types in multiple species and is therefore proving to be a much more powerful tool than other methods, such as polarized light microscopy, to evaluate tendon health and collagen quality [5].…”