Charles-André Couture

Imaging and modeling collagen architecture from the nano to micro scale

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.

Imaging the noncentrosymmetric structural organization of tendon with Interferometric Second Harmonic Generation microscopy.

We image the relative orientation of organized groups of noncentrosymmetric molecules (like collagen or myosin) at the micron scale in biological tissues by combining interferometry and Second Harmonic Generation (SHG) microscopy.

The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy

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.

Imaging the bipolarity of myosin filaments with Interferometric Second Harmonic Generation microscopy.

We report that combining interferometry with Second Harmonic Generation (SHG) microscopy provides valuable information about the relative orientation of noncentrosymmetric structures composing tissues. This is confirmed through the imaging of rat medial gastrocnemius muscle. The inteferometric Second Harmonic Generation (ISHG) images reveal that each side of the myosin filaments composing the A band of the sarcomere generates π phase shifted SHG signal which implies that the myosin proteins at each end of the filaments are oriented in opposite directions. This highlights the bipolar structural organization of the myosin filaments and shows that muscles can be considered as a periodically poled biological structure.

Femoral epiphyseal cartilage matrix changes at predilection sites of equine osteochondrosis: Quantitative MRI, second‐harmonic microscopy and histological findings

Osteochondrosis is an ischemic chondronecrosis of epiphyseal growth cartilage that results in focal failure of endochondral ossification and osteochondritis dissecans at specific sites in the epiphyses of humans and animals, including horses. The upstream events leading to the focal ischemia remain unknown. The epiphyseal growth cartilage matrix is composed of proteoglycan and collagen macromolecules and encases its vascular tree in canals. The matrix undergoes major dynamic changes in early life that could weaken it biomechanically and predispose it to focal trauma and vascular failure. Subregions in neonatal foal femoral epiphyses (n = 10 osteochondrosis predisposed; n = 6 control) were assessed for proteoglycan and collagen structure/content employing 3T quantitative MRI (3T qMRI: T1ρ and T2 maps). Site‐matched validations were made with histology, immunohistochemistry, and second‐harmonic microscopy. Growth cartilage T1ρ and T2 relaxation times were significantly increased (p < 0.002) within the proximal third of the trochlea, a site predisposed to osteochondrosis, when compared with other regions. However, this was observed in both control and osteochondrosis predisposed specimens. Microscopic evaluation of this region revealed an expansive area with low proteoglycan content and a hypertrophic‐like appearance on second‐harmonic microscopy. We speculate that this matrix structure and composition, though physiological, may weaken the epiphyseal growth cartilage biomechanically in focal regions and could enhance the risk of vascular failure with trauma leading to osteochondrosis. However, additional investigations are now required to confirm this. 3T qMRI will be useful for future non‐invasive longitudinal studies to track the osteochondrosis disease trajectory in animals and humans.

Probing microtubules polarity in mitotic spindles in situ using Interferometric Second Harmonic Generation Microscopy

The polarity of microtubules is thought to be involved in spindle assembly, cytokinesis or active molecular transport. However, its exact role remains poorly understood, mainly because of the challenge to measure microtubule polarity in intact cells. We report here the use of fast Interferometric Second Harmonic Generation microscopy to study the polarity of microtubules forming the mitotic spindles in a zebrafish embryo. This technique provides a powerful tool to study mitotic spindle formation and may be directly transferable for investigating the kinetics and function of microtubule polarity in other aspects of subcellular motility or in native tissues.

Harmonic Generation in Graphene and Carbon Nanotubes

Here, we report experimental observations of non-perturbative harmonic generation from monolayer and few-layer graphene, and from thin films of aligned semiconducting carbon nanotubes. We investigate the dependence of harmonic generation on polarization of driving field.

High Contrast Interferometric Second Harmonic Generation Microscopy Probes the Polarity of Fibrils in Complex Collagenous Tissues

We implemented for the first time Interferometric SHG microscopy, using femtosecond pulses, to image the relative fibrils polarity in cartilage and show that SHG intensity depends on the local ratio of fibrils with opposite polarities.