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.
According to previous studies, the nonlinear susceptibility tensor ratio χ /χ 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 χ /χ -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.
BackgroundArticular 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.ResultsThe 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.ConclusionThe 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.
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.
The second harmonic generation from collagen is highly sensitive to what extent collagen molecules are ordered into fibrils as the SHG signal is approximately proportional to the square of the fibril thickness. This can be problematic when interpreting SHG images as thick fibers are much brighter than thinner fibers such that quantification of the amount of collagen present is difficult. On the other hand SHG is therefore also a very sensitive probe to determine whether collagen have assembled into fibrils or are still dissolved as individual collagen molecules. This information is not available from standard histology or immunohistochemical techniques. The degree for fibrillation is an essential component for proper tissue function. We will present the usefulness of SHG imaging in tissue engineering of cartilage as well as cartilage related pathologies. When engineering cartilage it is essential to have the appropriate culturing conditions which cause the collagen molecules to assemble into fibrils. By employing SHG imaging we have studied how cell seeding densities affect the fibrillation of collagen molecules. Furthermore we have used SHG to study pathologies in developing cartilage in a porcine model. In both cases SHG reveals information which is not visible in conventional histology or immunohistochemistry.
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.
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.
Articular cartilage is located at the bone ends in synovial joints and enables smooth and pain‐free joint motion. Its function is in large part determined by a network of collagen fibrils organized into zones and compartments, but little is known about how this organization is achieved in growing animals [1]. The articular cartilage develops superficially to a layer of temporary growth cartilage. The growth cartilage is responsible for the expansion of the bone ends and is replaced by bone during growth. The interface between the immature articular cartilage and the growth cartilage is histologically indistinguishable [2]. On the other hand, aligned collagen fibrils encapsulate cells in the articular cartilage but not in the growth cartilage, so the immature articular cartilage and the growth cartilage can be distinguished by locating these fibrils with transmission electron microscopy (TEM) in growing animals [1]. However, TEM requires extensive sample preparation and has limited field of view. Second harmonic generation (SHG) microscopy is a promising image modality that is highly specific to collagen fibrils without any staining [3]. SHG is a coherent nonlinear optical process that is specific for non‐centrosymmetric structures such as collagen fibrils and is characterized by frequency‐doubling of light. In contrast to fluorescence, the SHG intensity and radiation direction is highly sensitive to the arrangement of collagen fibrils. In addition, as the arrangement of fibrils can vary at different length scales, this sensitiveness can depend on the size of the focal volume. The aim of this study was to examine if the aligned fibrils encapsulating cells could be visualized by SHG microscopy without any staining in order to locate the immature articular cartilage of a growing animal. The cartilage located at the distal femur of growing piglets was imaged by detecting the forward and backward directed SHG. Focusing objectives with different numerical aperture (NA), i.e. focal volume, were used to examine the collagen fibrils at different length scales. The results demonstrated that the ratio of forward to backward SHG was dependent on the NA and that the dependence on the NA varied with distance from the articular surface (Figure 1). The high forward to backward ratio at low NA near the articular surface (Figure 2) was interpreted to represent the aligned fibrils characterizing the articular cartilage. The gradual transition between the immature articular cartilage and the growth cartilage was therefore clearly visualized by SHG, and SHG microscopy is therefore a promising technique to study the development of the collagen matrix in articular cartilage.
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