Second harmonic generation imaging in tissue engineering and cartilage pathologies

Abstract

The second harmonic generation from collagen is highly sensitive to what extent collagen molecules are ordered into fibrils as the SHG signal is approximately proportional to the square of the fibril thickness. This can be problematic when interpreting SHG images as thick fibers are much brighter than thinner fibers such that quantification of the amount of collagen present is difficult. On the other hand SHG is therefore also a very sensitive probe to determine whether collagen have assembled into fibrils or are still dissolved as individual collagen molecules. This information is not available from standard histology or immunohistochemical techniques. The degree for fibrillation is an essential component for proper tissue function. We will present the usefulness of SHG imaging in tissue engineering of cartilage as well as cartilage related pathologies. When engineering cartilage it is essential to have the appropriate culturing conditions which cause the collagen molecules to assemble into fibrils. By employing SHG imaging we have studied how cell seeding densities affect the fibrillation of collagen molecules. Furthermore we have used SHG to study pathologies in developing cartilage in a porcine model. In both cases SHG reveals information which is not visible in conventional histology or immunohistochemistry.

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.…”

Multimodal imaging of harmonophores and application of high content imaging for early cancer detection

“…The figure below elucidates how different imaging techniques used for identifying specific biochemical or structural signatures, such as collagen detected by SHG microscopy, CARS showing the fat reservoirs in the cancer tissue can be linked together to provide a holistic view of how malignant tissues metabolize nutrients and later metastasize. These studies further made the case for high content imaging, to be popularized all over the world, in order to provide an early detection system for spread of cancer tissue [17,18,19] As demonstrated for BCC, dermal tissues were one of the first to get imaged using SHG due to the ease of access and availability of samples [20,21]. Chen and his group, showed SHG and TPEF as a viable hybrid imaging tool [22], by simultaneously imaging collagen and elastin respectively.…”

“…It was clear the isotropic nature of collagen fibers in keloids matched well with the visual observations made previously by Su et al in 2011. [21,23,25,26,27] The success of SHG imaging spurred interest in the early detection of breast cancer.…”