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1. Second harmonic generation characterization of collagen in whole bone

   https://doi.org/10.1364/BOE.391866  

   Pendleton, Emily G. ; Tehrani, Kayvan F. ; Barrow, Ruth P. ; Mortensen, Luke J. ( July 2020 , Biomedical Optics Express)

   Bone is a unique biological composite material made up of a highly structured collagen mesh matrix and mineral deposits. Although mineral provides stiffness, collagen’s secondary organization provides a critical role in bone elasticity. Here, we performed polarimetric analysis of bone collagen fibers using second harmonic generation (SHG) imaging to evaluate lamella sheets and collagen fiber integrity in intact cranial bone. Our polarimetric data was fitted to a model accounting for diattenuation, polarization cross-talk, and birefringence. We compared our data to the fitted model and found no significant difference between our polarimetric observation and the representation of these scattering properties up to 70µm deep. We also observed a loss of resolution as we imaged up to 70µm deep into bone but a conservation of polarimetric response. Polarimetric SHG allows for the discrimination of collagen lamellar sheet structures in intact bone. Our work could allow for label-free identification of disease states and monitor the efficacy of therapies for bone disorders.
2. Collagen Fibril Abnormalities in Human and Mice Abdominal Aortic Aneurysm

   Jones, B. ; Tonniges, J. R. ; Debsk, A.i ; Albert, B. ; Yeung, D. A. ; Gadde, N. ; Mahajan, A. ; Sharma, N. ; Calomeni, E. P. ; Go, M. ; et al ( August 2020 , Biomedical Engineering Society 2020 Annual Meeting)

   Introduction: Vascular diseases like abdominal aortic aneurysms (AAA) are characterized by a drastic remodeling of the vessel wall, accompanied with changes in the elastin and collagen content. At the macromolecular level, the elastin fibers in AAA have been reported to undergo significant structural alterations. While the undulations (waviness) of the collagen fibers is also reduced in AAA, very little is understood about changes in the collagen fibril at the sub-fiber level in AAA as well as in other vascular pathologies. Materials and Methods: In this study we investigated structural changes in collagen fibrils in human AAA tissue extracted at the time of vascular surgery and in aorta extracted from angiotensin II (AngII) infused ApoE−/− mouse model of AAA. Collagen fibril structure was examined using transmission electron microscopy and atomic force microscopy. Images were analyzed to ascertain length and depth of D-periodicity, fibril diameter and fibril curvature. Tissues were also stained using collagen hybridizing peptide (CHP) and analyzed using fluorescent microscopy and second harmonic generation (SHG) microscopy to locate regions of healthy and degraded collagen. Results: Abnormal collagen fibrils with compromised D-periodic banding were observed in the excised human tissue and in remodeled regions of AAA in AngII infused mice (Figuremore »1). These abnormal fibrils were characterized by statistically significant reduction in depths of D-periods and an increased curvature of collagen fibrils. These features were more pronounced in human AAA as compared to murine samples. Additionally, regions of abnormal collagen were located within the remodeled areas of AAA tissue and were distinct from healthy collagen regions as ascertained using CHP staining and SHG (Figure 1). Thoracic aorta from Ang II-infused mice, abdominal aorta from saline-infused mice, and abdominal aorta from non-AAA human controls did not contain abnormal collagen fibrils. Conclusions: The structural alterations in abnormal collagen fibrils appear similar to those reported for collagen fibrils subjected to mechanical overload or chronic inflammation in other tissues. Detection of abnormal collagen could be utilized to better understand the functional properties of the underlying extracellular matrix in vascular as well as other pathologies.« less
3. Ex-vivo quantification of ovine pia arachnoid complex biomechanical properties under uniaxial tension

   https://doi.org/10.1186/s12987-020-00229-w  

   Conley Natividad, Gabryel ; Theodossiou, Sophia K. ; Schiele, Nathan R. ; Murdoch, Gordon K. ; Tsamis, Alkiviadis ; Tanner, Bertrand ; Potirniche, Gabriel ; Mortazavi, Martin ; Vorp, David A. ; Martin, Bryn A. ( December 2020 , Fluids and Barriers of the CNS)

   Abstract Background The pia arachnoid complex (PAC) is a cerebrospinal fluid-filled tissue conglomerate that surrounds the brain and spinal cord. Pia mater adheres directly to the surface of the brain while the arachnoid mater adheres to the deep surface of the dura mater. Collagen fibers, known as subarachnoid trabeculae (SAT) fibers, and microvascular structure lie intermediately to the pia and arachnoid meninges. Due to its structural role, alterations to the biomechanical properties of the PAC may change surface stress loading in traumatic brain injury (TBI) caused by sub-concussive hits. The aim of this study was to quantify the mechanical and morphological properties of ovine PAC. Methods Ovine brain samples (n = 10) were removed from the skull and tissue was harvested within 30 min post-mortem. To access the PAC, ovine skulls were split medially from the occipital region down the nasal bone on the superior and inferior aspects of the skull. A template was used to remove arachnoid samples from the left and right sides of the frontal and occipital regions of the brain. 10 ex-vivo samples were tested with uniaxial tension at 2 mm s −1 , average strain rate of 0.59 s −1 , until failure at < 5 h post extraction. The force and displacement datamore »were acquired at 100 Hz. PAC tissue collagen fiber microstructure was characterized using second-harmonic generation (SHG) imaging on a subset of n = 4 stained tissue samples. To differentiate transverse blood vessels from SAT by visualization of cell nuclei and endothelial cells, samples were stained with DAPI and anti-von Willebrand Factor, respectively. The Mooney-Rivlin model for average stress–strain curve fit was used to model PAC material properties. Results The elastic modulus, ultimate stress, and ultimate strain were found to be 7.7 ± 3.0, 2.7 ± 0.76 MPa, and 0.60 ± 0.13, respectively. No statistical significance was found across brain dissection locations in terms of biomechanical properties. SHG images were post-processed to obtain average SAT fiber intersection density, concentration, porosity, tortuosity, segment length, orientation, radial counts, and diameter as 0.23, 26.14, 73.86%, 1.07 ± 0.28, 17.33 ± 15.25 µm, 84.66 ± 49.18°, 8.15%, 3.46 ± 1.62 µm, respectively. Conclusion For the sizes, strain, and strain rates tested, our results suggest that ovine PAC mechanical behavior is isotropic, and that the Mooney-Rivlin model is an appropriate curve-fitting constitutive equation for obtaining material parameters of PAC tissues.« less
4. Polarization Sensitive Photothermal Mid-Infrared Spectroscopic Imaging of Human Bone Marrow Tissue

   https://doi.org/10.1177/00037028211063513  

   Mankar, Rupali ; Gajjela, Chalapathi C. ; Bueso-Ramos, Carlos E. ; Yin, C. Cameron ; Mayerich, David ; Reddy, Rohith K. ( March 2022 , Applied Spectroscopy)

   Collagen quantity and integrity play an important role in understanding diseases such as myelofibrosis (MF). Label-free mid-infrared spectroscopic imaging (MIRSI) has the potential to quantify collagen while minimizing the subjective variance observed with conventional histopathology. Infrared (IR) spectroscopy with polarization sensitivity provides chemical information while also estimating tissue dichroism. This can potentially aid MF grading by revealing the structure and orientation of collagen fibers. Simultaneous measurement of collagen structure and biochemical properties can translate clinically into improved diagnosis and enhance our understanding of disease progression. In this paper, we present the first report of polarization-dependent spectroscopic variations in collagen from human bone marrow samples. We build on prior work with animal models and extend it to human clinical biopsies with a practical method for high-resolution chemical and structural imaging of bone marrow on clinical glass slides. This is done using a new polarization-sensitive photothermal mid-infrared spectroscopic imaging scheme that enables sample and source independent polarization control. This technology provides 0.5 µm spatial resolution, enabling the identification of thin (≈1 µm) collagen fibers that were not separable using Fourier Transform Infrared (FT-IR) imaging in the fingerprint region at diffraction-limited resolution ( ≈ 5 µm). Finally, we propose quantitative metrics to identify fiber orientation frommore »discrete band images (amide I and amide II) measured under three polarizations. Previous studies have used a pair of orthogonal polarization measurements, which is insufficient for clinical samples since human bone biopsies contain collagen fibers with multiple orientations. Here, we address this challenge and demonstrate that three polarization measurements are necessary to resolve orientation ambiguity in clinical bone marrow samples. This is also the first study to demonstrate the ability to spectroscopically identify thin collagen fibers (≈1 µm diameter) and their orientations, which is critical for accurate grading of human bone marrow fibrosis.

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5. Depth-resolved Mueller matrix polarimetry microscopy of the rat cornea

   https://doi.org/10.1364/BOE.402201  

   Du Le, V. N. ; Saytashev, Ilyas ; Saha, Sudipta ; Lopez, Pedro F. ; Laughrey, Megan ; Ramella-Roman, Jessica C. ( September 2020 , Biomedical Optics Express)

   Mueller matrix polarimetry (MMP) is a promising linear imaging modality that can enable visualization and measurement of the polarization properties of the cornea. Although the distribution of corneal birefringence has been reported, depth resolved MMP imaging of the cornea has not been archived and remains challenging. In this work, we perform depth-resolved imaging of the cornea using an improved system that combines Mueller matrix reflectance and transmission microscopy together with nonlinear microscopy utilizing second harmonic generation (SHG) and two photon excitation fluorescence (TPEF). We show that TPEF can reveal corneal epithelial cellular network while SHG can highlight the presence of corneal stromal lamellae. We then demonstrate that, in confocal reflectance measurement, as depth increases from 0 to 80μm both corneal depolarization and retardation increase. Furthermore, it is shown that the spatial distribution of corneal depolarization and retardation displays similar complexity in both reflectance (confocal and non-confocal) and transmission measurement, likely due to the strong degree of heterogeneity in the stromal lamellae.