Collagen fiber orientations in bones, visible with Second Harmonic Generation (SHG) microscopy, represent the inner structure and its alteration due to influences like cancer. While analyses of these orientations are valuable for medical research, it is not feasible to analyze the needed large amounts of local orientations manually. Since we have uncertain borders for these local orientations only rough regions can be segmented instead of a pixel-wise segmentation. We analyze the effect of these uncertain borders on human performance by a user study. Furthermore, we compare a variety of 2D and 3D methods such as classical approaches like Fourier analysis with state-of-the-art deep neural networks for the classification of local fiber orientations. We present a general way to use pretrained 2D weights in 3D neural networks, such as Inception-ResNet-3D a 3D extension of Inception-ResNet-v2. In a 10 fold cross-validation our two stage segmentation based on Inception-ResNet-3D and transferred 2D ImageNet weights achieves a human comparable accuracy.
Collagen structures in SHG images have been analyzed in several publications @cite_19 @cite_10 @cite_18 @cite_21 @cite_9 @cite_1 @cite_6 . They were analyzed in tissue @cite_19 and bones @cite_18 @cite_10 . @cite_1 presented how Fourier analysis can be used to investigate the orientation of collagen fibers. The Fourier analysis was extended from small regions to the whole scan in @cite_21 @cite_9 @cite_6 . The analysis classified small image parts as anisotropic, isotropic and dark. These classifications where used to calculate the distributions of classes over an image. In @cite_9 these distributions where used to detect injured tendons. Moreover, @cite_21 showed the change of distribution due to aging can be used to determine the age of pigs. @cite_6 used the 3D information of SHG data and could show an increase in performance.
@cite_18 @cite_9 @cite_21 @cite_1 @cite_6 @cite_19 @cite_10
2621852502 2017323918 1980068389 2056254464 2016266030 2077572701 2766462831
Interfaces provide the structural basis of essential bone functions. In the hierarchical structure of bone tissue, heterogeneities such as porosity or boundaries are found at scales ranging from nanometers to millimeters, all of which contributing to macroscopic properties. To date, however, the complexity or limitations of currently used imaging methods restrict our understanding of this functional integration. Here we address this issue using label-free third-harmonic generation (THG) microscopy. We find that the porous lacuno-canalicular network (LCN), revealing the geometry of osteocytes in the bone matrix, can be directly visualized in 3D with submicron precision over millimetric fields of view compatible with histology. THG also reveals interfaces delineating volumes formed at successive remodeling stages. Finally, we show that the structure of the LCN can be analyzed in relation with that of the extracellular matrix and larger-scale structures by simultaneously recording THG and second-harmonic generation (SHG) signals relating to the collagen organization. Fourier transform-second harmonic generation (FT-SHG) imaging is used as a technique for evaluating collagenase-induced injury in horse tendons. The differences in collagen fiber organization between normal and injured tendon are quantified. Results indicate that the organization of collagen fibers is regularly oriented in normal tendons and randomly organized in injured tendons. This is further supported through the use of additional metrics, in particular, the number of dark (no minimal signal) and isotropic (no preferred fiber orientation) regions in the images, and the ratio of forward-to-backward second-harmonic intensity. FT-SHG microscopy is also compared with the conventional polarized light microscopy and is shown to be more sensitive to assessing injured tendons than the latter. Moreover, sample preparation artifacts that affect the quantitative evaluation of collagen fiber organization can be circumvented by using FT-SHG microscopy. The technique has potential as an assessment tool for evaluating the impact of various injuries that affect collagen fiber organization. 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: 1 month, 3.5 months, 6 months, and 30 months, 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. Fourier transform-second-harmonic generation imaging is employed to obtain quantitative metrics of collagen fibers in biological tissues. In particular, the preferred orientation and maximum spatial frequency of collagen fibers for selected regions of interest in porcine trachea, ear, and cornea are determined. These metrics remain consistent when applied to collagen fibers in the ear, which can be expected from observation. Collagen fibers in the trachea are more random with large standard deviations in orientation, and large variations in maximum spatial frequency. In addition, these metrics are used to investigate structural changes through a 3D stack of the cornea. This technique can be used as a quantitative marker to assess the structure of collagen fibers that may change due to damage from disease or physical injury. We present three-dimensional Fourier transform-second-harmonic generation (3D FT-SHG) imaging, a generalization of the previously reported two-dimensional FT-SHG, to quantify collagen fiber organization from 3D image stacks of biological tissues. The current implementation calculates 3D preferred orientation of a region of interest, and classifies regions of interest based on orientation anisotropy and average voxel intensity. Presented are some example applications of the technique which reveal the layered structure of collagen fibers in porcine sclera, and estimates the cut angle of porcine tendon tissues. This technique shows promising potential for studying biological tissues that contain fibrillar structures in 3D. Although the nonlinear optical effect known as second-harmonic generation (SHG) has been recognized since the earliest days of laser physics and was demonstrated through a microscope over 25 years ago, only in the past few years has it begun to emerge as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. Only small modifications are required to equip a standard laser-scanning two-photon microscope for second-harmonic imaging microscopy (SHIM). Recent studies of the three-dimensional in vivo structures of well-ordered protein assemblies, such as collagen, microtubules and muscle myosin, are beginning to establish SHIM as a nondestructive imaging modality that holds promise for both basic research and clinical pathology. Thus far the best signals have been obtained in a transmitted light geometry that precludes in vivo measurements on large living animals. This drawback may be addressed through improvements in the collection of SHG signals via an epi-illumination microscope configuration. In addition, SHG signals from certain membrane-bound dyes have been shown to be highly sensitive to membrane potential. Although this indicates that SHIM may become a valuable tool for probing cell physiology, the small signal size would limit the number of photons that could be collected during the course of a fast action potential. Better dyes and optimized microscope optics could ultimately lead to the imaging of neuronal electrical activity with SHIM. Abstract 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.