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.

In solids, high harmonic radiation arises from the sub-cycle dynamics of electrons and holes under the action of an intense laser field. The strong field regime opens new opportunities to understand and control carrier dynamics on ultrafast time scales, including the coherent dynamics of quasiparticles such as massless Dirac fermions. Here, we irradiate monolayer and few-layer graphene with intense infrared light to produce non-perturbative harmonics of the fundamental up to the 7th order. We find that the polarization dependence shows surprising agreement with gas phase harmonics. Using a two-band model, we explore the nonlinear current due to electrons near the Dirac points, and we discuss the interplay between intraband and interband contributions to the harmonic spectrum. This interplay opens new opportunities to access ultrafast and strong-field physics of graphene.

Journal of BIOPHOTONICSWe report the imaging of tendon with Interferometric Second Harmonic Generation microscopy. We observe that the noncentrosymmetric structural organization can be maintained along the fibrillar axis over more than 150 mm, while in the transverse direction it is $1-15 mm. Those results are explained by modeling tendon as a heterogeneous distribution of noncentrosymmetric nanocylinders (collagen fibrils) oriented along the fibrillar axis. The preservation of the noncentrosymmetric structural organization over multiple tens of microns reveals that tendon is made of domains in which the ratio between fibrils with positive and negative polarity is unbalanced.A cross section image representing a laser beam focussed in connective tissue. Collagen fibrils appear as cylinders with a random positive or negative polarity (shown with red or green). The laser intensity profile shows the size of the focal spot.

In this work, we report the implementation of interferometric second harmonic generation (SHG) microscopy with femtosecond pulses. As a proof of concept, we imaged the phase distribution of SHG signal from the complex collagen architecture of juvenile equine growth cartilage. The results are analyzed in respect to numerical simulations to extract the relative orientation of collagen fibrils within the tissue. Our results reveal large domains of constant phase together with regions of quasi-random phase, which are correlated to respectively high- and low-intensity regions in the standard SHG images. A comparison with polarization-resolved SHG highlights the crucial role of relative fibril polarity in determining the SHG signal intensity. Indeed, it appears that even a well-organized noncentrosymmetric structure emits low SHG signal intensity if it has no predominant local polarity. This work illustrates how the complex architecture of noncentrosymmetric scatterers at the nanoscale governs the coherent building of SHG signal within the focal volume and is a key advance toward a complete understanding of the structural origin of SHG signals from tissues.

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.