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Abstract Extracellular matrix (ECM) represents more than a mere intercellular cement. It is physiologically active in cell communication, adhesion and proliferation. Collagen is the most abundant protein, making up to 90% of ECM, and 30% of total protein weight in humans. Second-harmonic generation (SHG) microscopy represents an important tool to study collagen organization of ECM in freshly unfixed tissues and paraffin-embedded tissue samples. This manuscript aims to review some of the applications of SHG microscopy in Oncologic Pathology, mainly in the study of ECM of epithelial tumors. It is shown how collagen parameters measured by this technique can aid in the differential diagnosis and in prognostic stratification. There is a tendency to associate higher amount, lower organization and higher linearity of collagen fibers with tumor progression and metastasizing. These represent complex processes, in which matrix remodeling plays a central role, together with cancer cell genetic modifications. Integration of studies on cancer cell biology and ECM are highly advantageous to give us a more complete picture of these processes. As microscopic techniques provide topographic information allied with biologic characteristics of tissue components, they represent important tools for a more complete understanding of cancer progression. In this context, SHG has provided significant insights in human tumor specimens, readily available for Pathologists.

Formation of the circular-shaped isolated domains surrounded by broad domain boundary (BDB) during dot ion beam irradiation in SBN:Ni crystals with submicron domain structure was studied. It was shown that domain and BDB sizes and depths increased proportionally to a square root of the irradiation dose. The radius decreased and BDB width increased with depth for dot and 2 D array exposure. The observed abnormal domain structure evolution was attributed to equiprobable positions of the steps due to merging with nanodomains. BDB increase with depth was explained by the features of spatial distribution of field polar component in the bulk. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD) is associated with the chronic progression of fibrosis. In general, the progression of liver fibrosis is determined by a histopathological assessment with a collagen-stained section; however, the ultra-early stage of liver fibrosis is challenging to identify because of the low sensitivity in the collagen-selective staining method. In the present study, we demonstrate the feasibility of second-harmonic generation (SHG) microscopy in the histopathological diagnosis of the liver of NAFLD patients for the quantitative assessment of the ultra-early stage of fibrosis. We investigated four representative NAFLD patients with early stages of fibrosis. SHG microscopy visualised well-matured fibrotic structures and early fibrosis diffusely involving liver tissues, whereas early fibrosis is challenging to be identified by conventional histopathological methods. Furthermore, the SHG emission directionality analysis revealed the maturation of each collagen fibre of each patient. As a result, SHG microscopy is feasible for assessing liver fibrosis on NAFLD patients, including the ultra-early stage of liver fibrosis that is difficult to diagnose by the conventional histopathological method. The assessment method of the ultra-early fibrosis by using SHG microscopy may serve as a crucial means for pathological, clinical, and prognostic diagnosis of NAFLD patients. [ABSTRACT FROM AUTHOR]

Our previous study on rat skin showed that cumulative oxidative pressure induces profound structural and ultrastructural alterations in both rat skin epidermis and dermis during aging. Here, we aimed to investigate the biophotonic properties of collagen as a main dermal component in the function of chronological aging. We used second harmonic generation (SHG) and two‐photon excited fluorescence (TPEF) on 5 μm thick skin paraffin sections from 15‐day‐, 1‐month‐ and 21‐month‐old rats, respectively, to analyze collagen alterations, in comparison to conventional light and electron microscopy methods. Obtained results show that polarization‐resolved SHG (PSHG) images can detect collagen fiber alterations in line with chronological aging and that this method is consistent with light and electron microscopy. Moreover, the β coefficient calculated from PSHG images points out that delicate alterations lead to a more ordered structure of collagen molecules due to oxidative damage. The results of this study also open the possibility of successfully applying this fast and label‐free method to previously fixed samples. [ABSTRACT FROM AUTHOR]

The rhythmic electrical activity of the heart’s natural pacemaker, the sinoatrial node (SAN), determines cardiac beating rate (BR). SAN electrical activity is tightly controlled by multiple factors, including tissue stretch, which may contribute to adaptation of BR to changes in venous return. In most animals, including human, there is a robust increase in BR when the SAN is stretched. However, the chronotropic response to sustained stretch differs in mouse SAN, where it causes variable responses, including decreased BR. The reasons for this species difference are unclear. They are thought to relate to dissimilarities in SAN electrophysiology (particularly action potential morphology) between mouse and other species and to how these interact with subcellular stretch-activated mechanisms. Furthermore, species-related differences in structural and mechanical properties of the SAN may influence the chronotropic response to SAN stretch. Here we assess (i) how the BR response to sustained stretch of rabbit and mouse isolated SAN relates to tissue stiffness, (ii) whether structural differences could account for observed differences in BR responsiveness to stretch, and (iii) whether pharmacological modification of mouse SAN electrophysiology alters stretch-induced chronotropy. We found disparities in the relationship between SAN stiffness and the magnitude of the chronotropic response to stretch between rabbit and mouse along with differences in SAN collagen structure, alignment, and changes with stretch. We further observed that pharmacological modification to prolong mouse SAN action potential plateau duration rectified the direction of BR changes during sustained stretch, resulting in a positive chronotropic response akin to that of other species. Overall, our results suggest that structural, mechanical, and background electrophysiological properties of the SAN influence the chronotropic response to stretch. Improved insight into the biophysical determinants of stretch effects on SAN pacemaking is essential for a comprehensive understanding of SAN regulation with important implications for studies of SAN physiology and its dysfunction, such as in the aging and fibrotic heart.

The rhythmic electrical activity of the heart's natural pacemaker, the sinoatrial node (SAN), determines cardiac beating rate (BR). SAN electrical activity is tightly controlled by multiple factors, including tissue stretch, which may contribute to adaptation of BR to changes in venous return. In most animals, including human, there is a robust increase in BR when the SAN is stretched. However, the chronotropic response to sustained stretch differs in mouse SAN, where it causes variable responses, including decreased BR. The reasons for this species difference are unclear. They are thought to relate to dissimilarities in SAN electrophysiology (particularly action potential morphology) between mouse and other species and to how these interact with subcellular stretch-activated mechanisms. Furthermore, species-related differences in structural and mechanical properties of the SAN may influence the chronotropic response to SAN stretch. Here we assess (i) how the BR response to sustained stretch of rabbit and mouse isolated SAN relates to tissue stiffness, (ii) whether structural differences could account for observed differences in BR responsiveness to stretch, and (iii) whether pharmacological modification of mouse SAN electrophysiology alters stretch-induced chronotropy. We found disparities in the relationship between SAN stiffness and the magnitude of the chronotropic response to stretch between rabbit and mouse along with differences in SAN collagen structure, alignment, and changes with stretch. We further observed that pharmacological modification to prolong mouse SAN action potential plateau duration rectified the direction of BR changes during sustained stretch, resulting in a positive chronotropic response akin to that of other species. Overall, our results suggest that structural, mechanical, and background electrophysiological properties of the SAN influence the chronotropic response to stretch. Improved insight into the biophysical determinants of stretch effects on SAN pacemaking is essential for a comprehensive understanding of SAN regulation with important implications for studies of SAN physiology and its dysfunction, such as in the aging and fibrotic heart. [ABSTRACT FROM AUTHOR]

Condensation is a fundamental property of mitotic chromosomes in eukaryotic cells. However, analyzing chromosome condensation in yeast is a challenging task while existing methods have notable weaknesses. Second-harmonic generation (SHG) microscopy is a label-free, advanced imaging technique for measuring the surface curve of isotropic molecules such as chromatin in live cells. We applied this method to detect changes in chromatin organization throughout the cell cycle in live yeast cells. We showed that SHG microscopy can be used to identify changes in chromatin organization throughout the cell cycle and in response to inactivation of the SMC complexes, cohesin and condensin. Implementation of this method will improve our ability to analyze chromatin structure in protozoa and will enhance our understanding of chromatin organization in eukaryotic cells. [ABSTRACT FROM AUTHOR]

Nanostructured conductive surfaces have been exploited for a variety of applications in photonics and optical sensing due to the broad spectral tunability of the localized surface plasmon resonances and the subsequent electromagnetic field confinement in the vicinity of the structures. Herein, we report on the second-order nonlinear optical properties of dendritic fractal structures with specific geometries that were optimized to yield resonances at both the fundamental and the second-harmonic wavelengths. Using a femtosecond excitation at 800 nm, second-harmonic generation microscopy experiments were conducted on a series of gold fractal dendritic structures with and without center of inversion. The polarized second-harmonic maps reveal the local character of the second-harmonic generation (SHG) emission and even structures with center of inversion show SHG activity in the periphery of the core dendron where non-centrosymmetric features are forming the higher fractal generations. The experiments were correlated using finite-difference time-domain electromagnetic time modelling performed at both the fundamental and second-harmonic wavelengths. [ABSTRACT FROM AUTHOR]

Polarization‐resolved second‐harmonic generation (P‐SHG) microscopy is a technique capable of characterizing nonlinear optical properties of noncentrosymmetric biomaterials by extracting the nonlinear susceptibility tensor components ratio χzzz2′/χzxx2′, with z‐axis parallel and x‐axis perpendicular to the C6 symmetry axis of molecular fiber, such as a myofibril or a collagen fiber. In this paper, we present two P‐SHG techniques based on incoming and outgoing circular polarization states for a fast extraction of χzzz2′/χzxx2′: A dual‐shot configuration where the SHG circular anisotropy generated using incident right‐ and left‐handed circularly‐polarized light is measured; and a single‐shot configuration for which the SHG circular anisotropy is measured using only one incident circular polarization state. These techniques are used to extract the χzzz2′/χzxx2′ of myosin fibrils in the body wall muscles of Drosophila melanogaster larva. The results are in good agreement with values obtained from the double Stokes‐Mueller polarimetry. The dual‐ and single‐shot circular anisotropy measurements can be used for fast imaging that is independent of the in‐plane orientation of the sample. They can be used for imaging of contracting muscles, or for high throughput imaging of large sample areas. [ABSTRACT FROM AUTHOR]

Achilles tendon rupture is a frequent injury with an increasing incidence. After clinical surgical repair, aimed at suturing the tendon stumps back into their original position, the repaired Achilles tendon is often plastically deformed and mechanically less strong than the pre-injured tissue, with muscle fatty degeneration contributing to function loss. Despite clinical outcomes, pre-clinical research has mainly focused on tendon structural repair, with a lack of knowledge regarding injury progression from tendon to muscle and its consequences on muscle degenerative/regenerative processes and function. Here, we characterize the morphological changes in the tendon, the myotendinous junction and muscle belly in a mouse model of Achilles tendon complete rupture, finding cellular and fatty infiltration, fibrotic tissue accumulation, muscle stem cell decline and collagen fiber disorganization. We use novel imaging technologies to accurately relate structural alterations in tendon fibers to pathological changes, which further explain the loss of muscle mechanical function after tendon rupture. The treatment of tendon injuries remains a challenge for orthopedics. Thus, the main goal of this study is to bridge the gap between clinicians’ knowledge and research to address the underlying pathophysiology of ruptured Achilles tendon and its consequences in the gastrocnemius. Such studies are necessary if current practices in regenerative medicine for Achilles tendon ruptures are to be improved.

Second-harmonic generation imaging (SHG) captures triple helical collagen molecules near tissue surfaces. Biomedical research routinely utilizes various imaging software packages to quantify SHG signals for collagen content and distribution estimates in modern tissue samples including bone. For the first time using SHG, samples of modern, medieval, and ice age bones were imaged to test the applicability of SHG to ancient bone from a variety of ages, settings, and taxa. Four independent techniques including Raman spectroscopy, FTIR spectroscopy, radiocarbon dating protocols, and mass spectrometry-based protein sequencing, confirm the presence of protein, consistent with the hypothesis that SHG imaging detects ancient bone collagen. These results suggest that future studies have the potential to use SHG imaging to provide new insights into the composition of ancient bone, to characterize ancient bone disorders, to investigate collagen preservation within and between various taxa, and to monitor collagen decay regimes in different depositional environments.

We investigate collective effects in plasmonic oligomers of different symmetries using second-harmonic generation (SHG) microscopy with cylindrical vector beams (CVBs). The oligomers consist of gold nanorods that have a longitudinal plasmon resonance close to the fundamental wavelength that is used for SHG excitation and whose long axes are arranged locally such that they follow the distribution of the transverse component of the electric field of radially or azimuthally polarized CVBs in the focal plane. We observe that SHG from such rotationally symmetric oligomers is strongly modified by the interplay between the polarization properties of the CVB and interparticle coupling. We find that the oligomers with radially oriented nanorods exhibit small coupling effects. In contrast, we find that the oligomers with azimuthally oriented nanorods exhibit large coupling effects that lead to silencing of SHG from the whole structure. Our experimental results are in very good agreement with numerical calculations based on the boundary element method. The work describes a new route for studying coupling effects in complex arrangements of nano-objects and thereby for tailoring the efficiency of nonlinear optical effects in such structures. [ABSTRACT FROM AUTHOR]

We present the first in situ 3D observation of domain wall dynamics close to a ferroelectric-paraelectric phase transition, which we obtain via second-harmonic generation microscopy (SHGM). Showing a pure second-order phase transition at its Curie temperature of 49 °C, triglycine sulfate (TGS) is a model ferroelectric material to study such phase transitions. After annealing, in TGS various qualitatively different domain patterns, e.g., stripes or small lenticular domains, have been reported so far. By applying SHGM, we can show that these findings do not necessarily contradict each other. In fact, qualitatively different domain patterns may coexist in the same TGS sample at the same time. [ABSTRACT FROM AUTHOR]

It is estimated that 2% to 4% of the US population will seek treatment for temporomandibular joint (TMJ) symptoms, typically occurring with anterior disc displacement. The temporomandibular retrodiscal tissue (RDT) has been postulated to restrict pathologic disc displacement. To elucidate RDT function, understanding regional RDT biomechanics and ultrastructure is required. No prior biomechanical analysis has determined regional variations in RDT properties or associated biomechanical outcomes with regional variations in collagen and elastin organization. The purpose of this study was to determine direction- and region-dependent tensile biomechanical characteristics and regional fibrillar arrangement of porcine RDT. Incremental stress relaxation experiments were performed on 20 porcine RDT specimens, with strain increments from 5% to 50%, a ramp-strain rate of 2% per second, and relaxation periods of 2.5 min. Tensile characteristics were determined between temporal and condylar regions and anteroposterior and mediolateral directions. RDT preparations were imaged using second-harmonic generation (SHG) microscopy for both collagen and elastin. Young's modulus showed significant differences by region ( P < 0.001) and strain ( P < 0.001). Young's modulus was <1 MPa from 5% to 20% strain, before increasing from 20% to 50% strain to a maximum of 2.9 MPa. Young's modulus trended higher in the temporal region and mediolateral direction. Instantaneous and relaxed moduli showed no significant difference by region or direction. Collagen arrangement was most organized near the disc boundary, with disorganization increasing posteriorly. Elastin was present at the disc boundary and RDT mid-body. Porcine RDT demonstrated region- and strain-dependent variations in tensile moduli, associated with regional differences in collagen and elastin. The small tensile moduli suggest that the RDT is not resistive to pathologic disc displacement. Further biomechanical analysis of the RDT is required to fully define RDT functional roles. Understanding regional variations in tissue stiffness and ultrastructure for TMJ components is critical to understanding joint function and for the long-term goal of improving TMJ disorder treatment strategies. [ABSTRACT FROM AUTHOR]

Tumor progression in breast cancer is significantly influenced by its interaction with the surrounding stromal tissue. Specifically, the composition, orientation, and alignment of collagen fibers in tumor-adjacent stroma affect tumor growth and metastasis. Most of the work done on measuring this prognostic marker has involved imaging of collagen fibers using second-harmonic generation microscopy (SHGM), which provides label-free specificity. Here, we show that spatial light interference microscopy (SLIM), a label-free quantitative phase imaging technique, is able to provide information on collagen-fiber orientation that is comparable to that provided by SHGM. Due to its wide-field geometry, the throughput of the SLIM system is much higher than that of SHGM and, because of the linear imaging, the equipment is simpler and significantly less expensive. Our results indicate that SLIM images can be used to extract important prognostic information from collagen fibers in breast tissue, potentially providing a convenient high throughput clinical tool for assessing patient prognosis. [ABSTRACT FROM AUTHOR]

We developed multimodal multiphoton microspectroscopy using a small-diameter probe with gradient-index lenses and applied it to unstained Alzheimer's disease (AD) brain samples. Our system maintained the image quality and spatial resolution of images obtained using an objective lens of similar numerical aperture. Multicolor images of AD brain samples were obtained simultaneously by integrating two-photon excited fluorescence and second-harmonic generation on a coherent anti-Stokes Raman scattering (CARS) microendoscope platform. Measurements of two hippocampal regions, the cornus ammonis-1 and dentate gyrus, revealed more lipids, amyloid fibers, and collagen in the AD samples than in the normal samples. Normal and AD brains were clearly distinguished by a large spectral difference and quantitative analysis of the CH mode using CARS micro-endoscope spectroscopy. We expect this system to be an important diagnosis tool in AD research. [ABSTRACT FROM AUTHOR]

Abstract: Injuries and damage to tendons plague both human and equine athletes. At the site of injuries, various cells congregate to repair and re-structure the collagen. Treatments for collagen injury range from simple procedures such as icing and pharmaceutical treatments to more complex surgeries and the implantation of stem cells. Regardless of the treatment, the level of mechanical stimulation incurred by the recovering tendon is crucial. However, for a given tendon injury, it is not known precisely how much of a load should be applied for an effective recovery. Both too much and too little loading of the tendon could be detrimental during recovery. A mapping of the complex local environment imparted to any cell present at the site of a tendon injury may however, convey fundamental insights related to their decision making as a function of applied load. Therefore, fundamentally knowing how cells translate mechanical cues from their external environment into signals regulating their functions during repair is crucial to more effectively treat these types of injuries. In this paper, we studied systems of tendons with a variety of 2-photon-based imaging techniques to examine the local mechanical environment of cells in both normal and injured tendons. These tendons were chemically treated to instigate various extents of injury and in some cases, were injected with stem cells. The results related by each imaging technique distinguish with high contrast and resolution multiple morphologies of the cells’ nuclei and the alignment of the collagen during injury. The incorporation of 2-photon FLIM into this study probed new features in the local environment of the nuclei that were not apparent with steady-state imaging. Overall, this paper focuses on horse tendon injury pattern and analysis with different 2-photon confocal modalities useful for wide variety of application in damaged tissues. [Copyright &y& Elsevier]

In this study we showed that second-harmonic generation (SHG) microscopy combined with precise methods for images evaluation can be used to detect structural changes in the human ovarian stroma. Using a set of scoring methods (alignment of collagen fibers, anisotropy, and correlation), we found significant differences in the distribution and organization of collagen fibers in the stroma component of serous, mucinous, endometrioid and mixed ovarian tumors as compared with normal ovary tissue. This methodology was capable to differentiate between cancerous and healthy tissue, with clear cut distinction between normal, benign, borderline, and malignant tumors of serous type. Our results indicated that the combination of different image-analysis approaches presented here represent a powerful tool to investigate collagen organization and extracellular matrix remodeling in ovarian tumors (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using β2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-ray minibeam. SHG imaging was found to provide about 2 µm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed. [ABSTRACT FROM AUTHOR]

Abstract: This review provides an introduction to second-harmonic generation (SHG) and an overview of zeolites and metal–organic frameworks as nonlinear optical materials. It furthermore demonstrates how SHG macro- and microscopy can be a powerful tool to probe adsorption, diffusion and organization of guest molecules in microporous materials. [Copyright &y& Elsevier]

Abstract: We propose the use of second-harmonic generation (SHG) microscopy for imaging collagen fibers in porcine femoral cortical bone. The technique is compared with scanning electron microscopy (SEM). SHG microscopy is shown to have excellent potential for bone imaging primarily due its intrinsic specificity to collagen fibers, which results in high contrast images without the need for specimen staining. Furthermore, this technique''s ability to quantitatively assess collagen fiber organization is evaluated through an exploratory examination of bone structure as a function of age, from very young to mature bone. In particular, four different age groups: 1month, 3.5months, 6months, and 30months, were studied. Specifically, we employ the recently developed Fourier transform-second harmonic generation (FT-SHG) imaging technique for the quantification of the structural changes, and observe that as the bone develops, there is an overall reduction in porosity, the number of osteons increases, and the collagen fibers become comparatively more organized. It is also observed that the variations in structure across the whole cross-section of the bone increase with age. The results of this work show that quantitative SHG microscopy can serve as a valuable tool for evaluating the structural organization of collagen fibers in ex vivo bone studies. [Copyright &y& Elsevier]

Achilles tendon rupture is a frequent injury with an increasing incidence. After clinical surgical repair, aimed at suturing the tendon stumps back into their original position, the repaired Achilles tendon is often plastically deformed and mechanically less strong than the pre-injured tissue, with muscle fatty degeneration contributing to function loss. Despite clinical outcomes, pre-clinical research has mainly focused on tendon structural repair, with a lack of knowledge regarding injury progression from tendon to muscle and its consequences on muscle degenerative/regenerative processes and function. Here, we characterize the morphological changes in the tendon, the myotendinous junction and muscle belly in a mouse model of Achilles tendon complete rupture, finding cellular and fatty infiltration, fibrotic tissue accumulation, muscle stem cell decline and collagen fiber disorganization. We use novel imaging technologies to accurately relate structural alterations in tendon fibers to pathological changes, which further explain the loss of muscle mechanical function after tendon rupture. The treatment of tendon injuries remains a challenge for orthopedics. Thus, the main goal of this study is to bridge the gap between clinicians' knowledge and research to address the underlying pathophysiology of ruptured Achilles tendon and its consequences in the gastrocnemius. Such studies are necessary if current practices in regenerative medicine for Achilles tendon ruptures are to be improved. [ABSTRACT FROM AUTHOR]

Collagen plays a key role in the strength of aortic walls, so studying micro-structural changes during disease development is critical to better understand collagen reorganization. Second-harmonic generation microscopy is used to obtain images of human aortic collagen in both healthy and diseased states. Methods are being developed in order to efficiently determine the waviness, that is, tortuosity and amplitude, as well as the diameter, orientation, and dispersion of collagen fibers, and bundles in healthy and aneurysmal tissues. The results show layer-specific differences in the collagen of healthy tissues, which decrease in samples of aneurysmal aortic walls. In healthy tissues, the thick collagen bundles of the adventitia are characterized by greater waviness, both in the tortuosity and in the amplitude, compared to the relatively thin and straighter collagen fibers of the media. In contrast, most aneurysmal tissues tend to have a more uniform structure of the aortic wall with no significant difference in collagen diameter between the luminal and abluminal layers. An increase in collagen tortuosity compared to the healthy media is also observed in the aneurysmal luminal layer. The data set provided can help improve related material and multiscale models of aortic walls and aneurysm formation.

Polarization‐dependent second‐harmonic generation (P‐SHG) microscopy is used to characterize molecular nonlinear optical properties of collagen and determine a three‐dimensional (3D) orientation map of collagen fibers within a pig tendon. C6 symmetry is used to determine the nonlinear susceptibility tensor components ratios in the molecular frame of reference χzzz2/χzxx2 and χxyz2/χzxx2, where the latter is a newly extracted parameter from the P‐SHG images and is related to the chiral structure of collagen. The χxyz2/χzxx2 is observed for collagen fibers tilted out of the image plane, and can have positive or negative values, revealing the relative polarity of collagen fibers within the tissue. The P‐SHG imaging was performed using a linear polarization‐in polarization‐out (PIPO) method on thin sections of pig tendon cut at different angles. The nonlinear chiral properties of collagen can be used to construct the 3D organization of collagen in the tissue and determine the orientation‐independent molecular susceptibility ratios of collagen fibers in the molecular frame of reference. Chirality of collagen is studied in a tendon tissue using a polarimetric second‐harmonic generation microscopy technique. A three‐dimensional (3D) orientation and the relative polarity of neighboring fibers are revealed. This information can be utilized for 3D reconstruction of collagen organization, which aid scientists in better understanding of the architecture and function of the collagen‐based tissues. [ABSTRACT FROM AUTHOR]

Recent evidence has implicated collagen, particularly fibrillar collagen, in a number of diseases ranging from osteogenesis imperfecta and asthma to breast and ovarian cancer. A key property of collagen that has been correlated with disease has been the alignment of collagen fibers. Collagen can be visualized using a variety of imaging techniques including second-harmonic generation (SHG) microscopy, polarized light microscopy, and staining with dyes or antibodies. However, there exists a great need to easily and robustly quantify images from these modalities for individual fibers in specified regions of interest and with respect to relevant boundaries. Most currently available computational tools rely on calculation of pixel-wise orientation or global window-wise orientation that do not directly calculate or give visible fiber-wise information and do not provide relative orientation against boundaries. We describe and detail how to use a freely available, open-source MATLAB software framework that includes two separate but linked packages "CurveAlign" and "CT-FIRE" that can address this need by either directly extracting individual fibers using an improved fiber tracking algorithm or directly finding optimal representation of fiber edges using the curvelet transform. This curvelet-based framework allows the user to measure fiber alignment on a global, region of interest, and fiber basis. Additionally, users can measure fiber angle relative to manually or automatically segmented boundaries. This tool does not require prior experience of programming or image processing and can handle multiple files, enabling efficient quantification of collagen organization from biological datasets.

The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by applying a quantitative image analysis technique for determining collagen fiber direction and density in second-harmonic generation microscopy images. This paper presents a collagen tissue analysis of scarred cheek pouches of four adult male Golden Syrian hamsters as an animal model for vocal fold scarring. One cheek pouch was scarred using an electrocautery unit and the other cheek was used as a control for each hamster. A home-built upright microscope and a compact ultrafast fiber laser were used to acquire depth resolved epi-collected second-harmonic generation images of collagen fibers. To quantify the average fiber direction and fiber density in each image, we applied two-dimensional Fourier analysis and intensity thresholding at five different locations for each control and scarred tissue sample, respectively. The resultant depth-resolved average fiber direction variance for scarred hamster cheek pouches (0.61 ± 0.03) was significantly lower (p < 0.05) than control tissue (0.73 ± 0.04), indicating increased fiber alignment within the scar. Depth-resolved average voxel density measurements indicated scarred tissues contained greater (p < 0.005) fiber density (0.72 ± 0.09) compared to controls (0.18 ± 0.03). In the present study, image analysis of both fiber alignment and density from depth-resolved second-harmonic generation images in epi-detection mode enabled the quantification of the increased collagen fiber deposition and alignment typically observed in fibrosis. The epi-detection geometry is the only viable method for in vivo imaging as well as imaging thick turbid tissues. These quantitative endpoints, clearly differentiating between control and scarred hamster cheek pouches, provide an objective means to characterize the extent of vocal fold scarring in vivo in preclinical and clinical research. In particular, this non-invasive method offers advantages for monitoring scar treatments in live animals and following the effects of scarring-related treatments such as application of steroids or drugs targeting pathways involved in fibrosis. SCANNING 38:684-693, 2016. © 2016 Wiley Periodicals, Inc., (© Wiley Periodicals, Inc.)

Characterization of collagen fiber angle distribution throughout the blood vessel wall provides insight into the mechanical behavior of healthy and diseased arteries and their capacity to remodel. Atherosclerotic plaque contributes to the overall mechanical behavior, yet little is known experimentally about how collagen fiber orientation is influenced by atherogenesis. We hypothesized that atherosclerotic lesion development, and the factors contributing to lesion development, leads to a shift in collagen fiber angles within the aorta. Second-harmonic generation microscopy was used to visualize the three-dimensional organization of collagen throughout the aortic wall and to examine structural differences in mice maintained on high-fat Western diet versus age-matched chow diet mice in a model of atherosclerosis. Image analysis was performed on thoracic and abdominal sections of the aorta from each mouse to determine fiber orientation, with the circumferential (0°) and blood flow directions (axial ±90°) as the two reference points. All measurements were used in a multiple regression analysis to determine the factors having a significant influence on mean collagen fiber angle. We found that mean absolute angle of collagen fibers is 43° lower in Western diet mice compared with chow diet mice. Mice on a chow diet have a mean collagen fiber angle of ±63°, whereas mice on a Western diet have a more circumferential fiber orientation (~20°). This apparent shift in absolute angle coincides with the development of extensive aortic atherosclerosis, suggesting that atherosclerotic factors contribute to collagen fiber angle orientation.