Stéphane Bancelin

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

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.

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.

Nonlinear optical properties of calcium barium niobate epitaxial thin films.

We investigate the potential of epitaxial calcium barium niobate (CBN) thin film grown by pulsed laser deposition for optical frequency conversion. Using second harmonic generation (SHG), we analyze the polarization response of the generated signal to determine the ratios d₁₅ / d₃₂ and d₃₃ / d₃₂ of the three independent components of the second-order nonlinear susceptibility tensor in CBN thin film. In addition, a detailed comparison to the signal intensity obtained in a y-cut quartz allows us to measure the absolute value of these components in CBN thin film: d₁₅ = 5 ± 2 pm / V, d₃₂ = 3.1 ± 0.6 pm / V and d₃₃ = 9 ± 2 pm / V.

In tendons, differing physiological requirements lead to functionally distinct nanostructures

The collagen-based tissues of animals are hierarchical structures: even tendon, the simplest collagenous tissue, has seven to eight levels of hierarchy. Tailoring tissue structure to match physiological function can occur at many different levels. We wanted to know if the control of tissue architecture to achieve function extends down to the nanoscale level of the individual, cable-like collagen fibrils. Using tendons from young adult bovine forelimbs, we performed stress-strain experiments on single collagen fibrils extracted from tendons with positional function, and tendons with energy storing function. Collagen fibrils from the two tendon types, which have known differences in intermolecular crosslinking, showed numerous differences in their responses to elongation. Unlike those from positional tendons, fibrils from energy storing tendons showed high strain stiffening and resistance to disruption in both molecular packing and conformation, helping to explain how these high stress tissues withstand millions of loading cycles with little reparative remodeling. Functional differences in load-bearing tissues are accompanied by important differences in nanoscale collagen fibril structure.

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.

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.