Analysis of forward and backward Second Harmonic Generation images to probe the nanoscale structure of collagen within bone and cartilage

Abstract

Collagen ultrastructure plays a central role in the function of a wide range of connective tissues. Studying collagen structure at the microscopic scale is therefore of considerable interest to understand the mechanisms of tissue pathologies. Here, we use second harmonic generation microscopy to characterize collagen structure within bone and articular cartilage in human knees. We analyze the intensity dependence on polarization and discuss the differences between Forward and Backward images in both tissues. Focusing on articular cartilage, we observe an increase in Forward/Backward ratio from the cartilage surface to the bone. Coupling these results to numerical simulations reveals the evolution of collagen fibril diameter and spatial organization as a function of depth within cartilage.

Tensile biomechanical properties of human temporomandibular joint disc: Effects of direction, region and sex

“…Second-harmonic generation microscopy, capable of imaging intact unstained collagen fiber orientation and organization (Chen et al, 2012; Houle et al, 2015; Lilledahl et al, 2015), revealed regional variations in human TMJ disc fiber orientation and organization, similar to that reported for porcine TMJ discs using polarized light microscopy and scanning electron microscopy (Detamore and Athanasiou, 2003; Shi et al, 2013). In the central region of the disc, collagen fibers run predominately in the anteroposterior direction and were tightly bound, supporting a primary direction of stretching in the anteroposterior direction during joint articulation.…”

“…To determine tissue microstructure, the superior surface of an intact human TMJ disc was imaged using second-harmonic generation microscopy (Chen et al, 2012; Houle et al, 2015; Lilledahl et al, 2015). The disc was dissected from a fresh-frozen 73-year-old female cadaver, frozen until use at −20 °C.…”

“…Regional variations in disc response were investigated with pairwise comparisons within each direction, and comparison of centrally located specimens in the anteroposterior and mediolateral directions. Regional collagen fiber orientation and organization were evaluated using second-harmonic generation microscopy, a significant advancement over traditional histological or imaging techniques, capable of imaging intact and unstained tissues (Chen et al, 2012; Houle et al, 2015; Lilledahl et al, 2015). Sex dependent biomechanical differences were anticipated, supported by sex specific differences in human temporomandibular disc electrical conductivity, porosity and ion diffusivity (Wright et al, 2013), potentially explaining the differences in the prevalence and severity of TMJDs among women.…”

Label-free imaging of bone multiscale porosity and interfaces using third-harmonic generation microscopy

“…SHG provides an intrinsic contrast specific to fibrillar collagen, in particular in the bone matrix 36–40 , and hence provides an image of the collagen matrix intrinsically registered with the THG image. The SHG signal depends on the degree of alignment and packing density of the collagen fibrils as well as their relative orientation with respect to the imaging plane and to the polarization of the excitation beam.…”

Modern trends in biophotonics for clinical diagnosis and therapy to solve unmet clinical needs

“…This methodology was capable to differentiate between cancerous and healthy tissue, with clear cut distinction between normal, benign, borderline, and malignant tumors of serous type. SHG was also applied to characterize collagen structure within bone and articular cartilage in human knees . This study analyzed the intensity dependence on polarization and discussed the differences between forward and backward images.…”