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.

Fast interferometric second harmonic generation microscopy.

We report the implementation of fast Interferometric Second Harmonic Generation (I-SHG) microscopy to study the polarity of non-centrosymmetric structures in biological tissues. Using a sample quartz plate, we calibrate the spatially varying phase shift introduced by the laser scanning system. Compensating this phase shift allows us to retrieve the correct phase distribution in periodically poled lithium niobate, used as a model sample.

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.

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.

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.

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.

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

We 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 μm, while in the transverse direction it is ∼1-15 μm. Those results are explained by modeling tendon as a heterogeneous distribution of noncentrosymmetric nano-cylinders (collagen fibrils) oriented along the fibrillar axis.