Analyzing the feasibility of discriminating between collagen types I and II using polarization-resolved second harmonic generation.

According to previous studies, the nonlinear susceptibility tensor ratio χ33 /χ31 obtained from polarization-resolved second harmonic generation (P-SHG) under the assumption of cylindrical symmetry can be used to distinguish between fibrillar collagen types. Discriminating between collagen fibrils of types I and II is important in tissue engineering of cartilage. However, cartilage has a random organization of collagen fibrils, and the assumption of cylindrical symmetry may be incorrect. In this study, we simulated the P-SHG response from different collagen organizations and demonstrated a possible method to exclude areas where cylindrical symmetry is not fulfilled and where fibrils are located in the imaging plane. The χ33 /χ31 -ratio for collagen type I in tendon and collagen type II in cartilage was estimated to be 1.33 and 1.36, respectively, using this method. These ratios are now much closer than what has been reported previously in the literature, and the larger reported differences between collagen types can be explained by variation in the structural organization.

Automated calibration and control for polarization-resolved second harmonic generation on commercial microscopes.

Polarization-resolved second harmonic generation (P-SHG) microscopy has evolved as a promising technique to reveal subresolution information about the structure and orientation of ordered biological macromolecules. To extend the adoption of the technique, it should be easily integrated onto commercial laser scanning microscopes. Furthermore, procedures for easy calibration and assessment of measurement accuracy are essential, and measurements should be fully automated to allow for analysis of large quantities of samples. In this paper we present a setup for P-SHG which is readily incorporated on commercial multiphoton microscopes. The entire system is completely automated which allows for rapid calibration through the freely available software and for automated imaging for different polarization measurements, including linear and circular polarization of the excitation beam. The results show that calibration settings are highly system dependent. We also show that the accuracy of the polarization control is easily quantified and that it varies between systems. The accuracy can be tuned by iterative alignment of optics or a more fine-grained calibration procedure. Images of real samples show that the red accuracy of the results is easily visualized with the automated setup. Through this system we believe that P-SHG could develop a wider adoption in biomedical applications.

Characterization of cellular and matrix alterations in the early pathogenesis of osteochondritis dissecans in pigs using second harmonic generation and two-photon excitation fluorescence microscopy.

Osteochondritis dissecans is a joint disease that is observed in several species. The disease can develop as a cause of ischemic chondronecrosis in the epiphyseal growth cartilage. Some lesions of chondronecrosis undergo spontaneous resolution, but it is not possible to predict whether a lesion will resolve or progress and require intervention. Proliferation of cells into clusters occurs at the lesion margin, but it is unclear if the clusters have a repair function. The aims of the current study were to examine clusters and potential matrix changes in response to ischemic chondronecrosis in the distal femur of 10 pigs aged 70-180 days using advanced microscopy based on two-photon excitation fluorescence and second harmonic generation. These microscopy techniques can perform 3D imaging of cells and collagen without staining. The results indicated a lower collagen density in the chondronecrotic areas compared to the normal growth cartilage, and fissures and breaks in the matrix integrity were demonstrated that potentially can propagate and cause osteochondritis dissecans. A higher number of cells in clusters was correlated with reduction in collagen density in the lesions. Some of the cells in the clusters had a morphology similar to progenitor cells, suggesting a potential repair role of the clusters. The study has shed further light on the secondary responses after initial lesion formation, which information can be of potential use to create models that can predict lesion progression and that may hence avoid unnecessary interventions in the future. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Non-linear optical microscopy of cartilage canals in the distal femur of young pigs may reveal the cause of articular osteochondrosis.

BACKGROUND: Articular osteochondrosis is a common cause of leg weakness in pigs and is defined as a focal delay in the endochondral ossification of the epiphysis. The first demonstrated steps in the pathogenesis consist of loss of blood supply and subsequent chondronecrosis in the epiphyseal growth cartilage. Blood vessels in cartilage are located in cartilage canals and become incorporated into the secondary ossification centre during growth. It has been hypothesized that vascular failure occurs during this incorporation process, but it is not known what predisposes a canal to fail. To obtain new information that may reveal the cause of vascular failure, the distal femur of 4 pigs aged 82-140 days was sampled and examined by non-linear optical microscopy. This novel technique was used for its ability to reveal information about collagen by second harmonic generation and cellular morphology by two-photon-excited fluorescence in thick sections without staining. The aims were to identify morphological variations between cartilage canal segments and to examine if failed cartilage canals could be followed back to the location where the blood supply ceased. RESULTS: The cartilage canals were shown to vary in their content of collagen fibres (112/412 segments), and the second harmonic and fluorescence signals indicated a variation in the bundling of collagen fibrils (245/412 segments) and in the calcification (30/412 segments) of the adjacent cartilage matrix. Failed cartilage canals associated with chondronecrosis were shown to enter the epiphyseal growth cartilage from not only the secondary ossification centre, but also the attachment site of the caudal cruciate ligament. CONCLUSION: The variations between cartilage canal segments could potentially explain why the blood supply fails at the osteochondral junction in only a subset of the canals. Proteins linked to these variations should be examined in future genomic studies. Although incorporation can still be a major cause, it could not account for all cases of vascular failure. The role of the caudal cruciate ligament in the cause of osteochondrosis should therefore be investigated further.

Second harmonic generation imaging reveals a distinct organization of collagen fibrils in locations associated with cartilage growth.

PURPOSE: The articular-epiphyseal cartilage complex (AECC) is responsible for the expansion of the bone ends and serves the function of the articular cartilage in juvenile mammals. Bundles of collagen fibrils surrounding cells were in the literature observed more frequently near the articular surface of the AECC. The articular surface, the perichondrium, and cartilage canals are interfaces where appositional growth of the AECC has been demonstrated. The current study aimed to evaluate the potential of second harmonic generation (SHG) to locate the collagen fibril bundles near the articular surface and to examine whether a comparable collagen fibril organization could be observed near the other interfaces of the AECC. MATERIALS AND METHODS: The study included the femoral condyle of four piglets aged 82-141 days. The forward and backward scattered SHG, and their ratio, was analyzed across the AECC using objectives with different numerical aperture. Two-photon-excited fluorescence was used to visualize cells. RESULTS: A similar pattern of collagen fibril organization was observed near the articular surface, around cartilage canals, and adjacent to the perichondrium. The pattern consisted of a higher ratio of forward to backward scattered SHG that increased relative to the surrounding matrix at lower numerical aperture. This was interpreted to reflect collagen fibril bundles in the territorial matrix of cells in these areas. CONCLUSIONS: The observed arrangement of collagen fibrils was suggested to be related to the presumed different growth activity in these areas and indicated that SHG may be used as an indirect and label-free marker for cartilage matrix growth.

Polarization second harmonic generation microscopy provides quantitative enhanced molecular specificity for tissue diagnostics.

Due to specific structural organization at the molecular level, several biomolecules (e.g., collagen, myosin etc.) which are strong generators of second harmonic generation (SHG) signals, exhibit unique responses depending on the polarization of the excitation light. By using the polarization second harmonic generation (p-SHG) technique, the values of the second order susceptibility components can be used to differentiate the types of molecule, which cannot be done by the use of a standard SHG intensity image. In this report we discuss how to implement p-SHG on a commercial multiphoton microscope and overcome potential artifacts in susceptibility (χ) image. Furthermore we explore the potential of p-SHG microscopy by applying the technique to different types of tissue in order to determine corresponding reference values of the ratio of second-order χ tensor elements. These values may be used as a bio-marker to detect any structural alterations in pathological tissue for diagnostic purposes. The SHG intensity image (red) in (a) shows the distribution of collagen fibers in ovary tissue but cannot determine the type of collagen fiber. However, the histogram distribution (b) for the values of the χ tensor element ratio can be used to quantitatively identify the types of collagen fibers.