Imaging and modeling collagen architecture from the nano to micro scale

Abstract

The collagen meshwork plays a central role in the functioning of a range of tissues including cartilage, tendon, arteries, skin, bone and ligament. Because of its importance in function, it is of considerable interest for studying development, disease and regeneration processes. Here, we have used second harmonic generation (SHG) to image human tissues on the hundreds of micron scale, and developed a numerical model to quantitatively interpret the images in terms of the underlying collagen structure on the tens to hundreds of nanometer scale. Focusing on osteoarthritic changes in cartilage, we have demonstrated that this combination of polarized SHG imaging and numerical modeling can estimate fibril diameter, filling fraction, orientation and bundling. This extends SHG microscopy from a qualitative to quantitative imaging technique, providing a label-free and non-destructive platform for characterizing the extracellular matrix that can expand our understanding of the structural mechanisms in disease.

Determination of collagen fibril size via absolute measurements of second-harmonic generation signals

“…Such an antiparallel organization was recently demonstrated in a direct way by the use of piezoelectric microscopy 33 . It results in partially destructive interferences in the forward direction and decreases the forward-detected SHG signal compared with the signal expected for parallel fibrils 5,27,[33][34][35] . The forward-detected SHG signal is then still larger than the backward-detected one, but the ratio of these signals is not as high as expected from the ratio of the coherence lengths in forward and backward directions, in agreement to measures in tissues such as tendons or fascia [35][36][37] .…”

“…Nevertheless, complementary approaches may be used to quantify dense collagen distributions in tissues, by combining numerical calculation and measurements sensitive to the collagen organization within the focal volume as recently reported 35 . Indeed, given a specific distribution of collagen fibrils within the focal volume (filling fraction, diameter and direction of fibrils), the overall SHG signal can be calculated as the coherent summation of the SHG response of each fibril and compared with polarization-resolved measurements, interferometric measurements or comparative forward/backward detection that provide additional information about collagen distribution at sub-micrometer scale 19,29,35,39,40, .…”

Nonlinear optical microscopy of early stage (ICRS Grade-I) osteoarthritic human cartilage

“…The physical origin and mathematical treatment of second harmonic generation (SHG) in biological tissues is described elsewhere [9,10]. Since, collagen is one of the major components of cartilage (~60% of dry weight), SHG microscopy can provide vital information about the structure and morphology of the extracellular matrix (ECM) of articular cartilage.…”

Articular cartilage zonal differentiation via 3D Second-Harmonic Generation imaging microscopy

“…Currently, there have only been a few studies using SHG on articular cartilage (27-32). Most of this work only utilized backward propagating SHG due to its ease of detection with minimal microscope modifications, whereas most of the signal is forward directed.…”

“…Additionally, many of these studies used thin human cartilage sections (~10-25 μm), embedded the tissue in paraffin, and imaged in the sagittal plane to characterize each zone as seen previously in the gold standard of histology. More recently, work from Dumas (35), Davies (30), and Légaré (32) have used forward SHG to model the collagen architecture of articular cartilage. Collectively, the results from these prior studies suggest that cartilage structure and orientation can be determined through SHG; however, structural aspects have not yet been fully determined (27-32).…”