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
1980068389 2016266030 2017323918 2766462831
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. 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. 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 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.