Imaging and 3D morphological analysis of collagen fibrils

The recent booming of multiphoton imaging of collagen fibrils by means of second harmonic generation microscopy generates the need for the development and automation of quantitative methods for image analysis. Standard approaches sequentially analyse two-dimensional (2D) slices to gain knowledge on the spatial arrangement and dimension of the fibrils, whereas the reconstructed three-dimensional (3D) image yields better information about these characteristics. In this work, a 3D analysis method is proposed for second harmonic generation images of collagen fibrils, based on a recently developed 3D fibre quantification method. This analysis uses operators from mathematical morphology. The fibril structure is scanned with a directional distance transform. Inertia moments of the directional distances yield the main fibre orientation, corresponding to the main inertia axis. The collaboration of directional distances and fibre orientation delivers a geometrical estimate of the fibre radius. The results include local maps as well as global distribution of orientation and radius of the fibrils over the 3D image. They also bring a segmentation of the image into foreground and background, as well as a classification of the foreground pixels into the preferred orientations. This accurate determination of the spatial arrangement of the fibrils within a 3D data set will be most relevant in biomedical applications. It brings the possibility to monitor remodelling of collagen tissues upon a variety of injuries and to guide tissues engineering because biomimetic 3D organizations and density are requested for better integration of implants.     

Image analysis of mineralized and non-mineralized type I collagen fibrils

Turkey leg tendons at an early stage of mineralization have been thin sectioned and imaged by electron microscopy. At this stage collagen-associated mineral apatite was found to be present within both the gap and overlap zones. The earliest apatite occurs in a microcrystalline form which gives a rather generalized and characteristic density to both the gap and overlap zones; with subsequent development larger defined apatite crystals arise which span gap/overlap zones. Fourier transformation of such images revealed the major 67 nm axial repeat of the gap/overlap zone plus four other maxima corresponding to repeat spacings of 22, 16, 13, and 11 nm respectively. Computer imaging techniques were used to reconstruct images by using selected spatial frequencies from such transforms. In this manner the subperiodic distributions of mineral were visually enhanced. These subperiodicities are positioned in an asymmetric fashion over the entire D unit repeat aligning with the molecular orientation of the fibril. Analyses of both negatively stained collagen and computer-generated maps of collagen hydrophobicity were compared to the mineral distribution of collagen. Densitometric comparisons showed a positional correlation between the axial banding patterns of mineralized fibrils and those of negatively stained non-mineralized fibrils. Comparable spatial frequencies were also present in transforms between hydrophobic maps and mineral distribution of collagen. These results suggest that the lateral clusterings of hydrophobic residues which span the fibril at specific sites in both the gap and overlap zones serve to prohibit early mineral deposition. This observed hydrophobic influence in combination with the gap space appear as contributing factors in the observed axial distribution of mineral within collagen.     

Label‐free detection of fibrillar collagen deposition associated with vascular elements in glioblastoma multiforme by using multiphoton microscopy

Glioblastoma multiforme (GBM‐WHO grade IV) is the most common and the most aggressive form of brain tumors in adults with the median survival of 10–12 months. The diagnostic detection of extracellular matrix (ECM) component in the tumour microenvironment is of prognostic value. In this paper, the fibrillar collagen deposition associated with vascular elements in GBM were investigated in the fresh specimens and unstained histological slices by using multiphoton microscopy (MPM) based on two‐photon excited fluorescence (TPEF) and second harmonic generation (SHG). Our study revealed the existence of fibrillar collagen deposition in the adventitia of remodelled large blood vessels and in glomeruloid vascular structures in GBM. The degree of fibrillar collagen deposition can be quantitatively evaluated by measuring the adventitial thickness of blood vessels or calculating the ratio of SHG pixel to the whole pixel of glomeruloid vascular structure in MPM images. These results indicated that MPM can not only be employed to perform a retrospective study in unstained histological slices but also has the potential to apply for in vivo brain imaging to understand correlations between malignancy of gliomas and fibrillar collagen deposition.     

Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging

The aim of this work is to demonstrate that multiphoton microscopy is a preferred technique to investigate intact cornea structure without slicing and staining. At the micron resolution, multiphoton imaging can provide both large morphological features and detailed structure of epithelium, corneal collagen fibril bundles and keratocytes. A large area multiphoton cross-section across an intact eye excised from a GFP mouse was obtained by a homebuilt multiphoton microscope. The broadband multiphoton fluorescence (435-700 nm) and second harmonic generation (SHG, 360-400 nm) signals were generated by the 760 nm output of a femtosecond titanium-sapphire laser. A water immersion objective (Fluor, 40X, NA 0.8; Nikon) was used to facilitate imaging the curve ocular surface. The multiphoton image over entire cornea provides morphological information of epithelial cells, keratocytes, and global collagen orientation. Specifically, our planar, large area multiphoton image reveals a concentric pattern of the stroma collagen, indicative of the laminar collagen organization throughout the stroma. In addition, the green fluorescence protein (GFP) labeling contributed to fluorescence contrast of cellular area and facilitated visualizing of inactive keratocytes. Our results show that multiphoton imaging of GFP labeled mouse cornea manifests both morphological significance and structural details. The second harmonic generation imaging reveals the collagen orientation, while the multiphoton fluorescence imaging indicates morphology and distribution of cells in cornea. Our results support that multiphoton microscopy is an appropriate technology for further in vivo investigation and diagnosis of cornea.     

TOC ‐ Issue Information

Knowledge of the collagen structure of an Achilles tendon is critical to comprehend the physiology, biomechanics, homeostasis and remodelling of the tissue. Despite intensive studies, there are still uncertainties regarding the microstructure. The majority of studies have examined the longitudinally arranged collagen fibrils as they are primarily attributed to the principal tensile strength of the tendon. Few studies have considered the structural integrity of the entire three‐dimensional (3D) collagen meshwork, and how the longitudinal collagen fibrils are integrated as a strong unit in a 3D domain to provide the tendons with the essential tensile properties. Using second harmonic generation imaging, a 3D imaging technique was developed and used to study the 3D collagen matrix in the midportion of Achilles tendons without tissue labelling and dehydration. Therefore, the 3D collagen structure is presented in a condition closely representative of the in vivo status. Atomic force microscopy studies have confirmed that second harmonic generation reveals the internal collagen matrix of tendons in 3D at a fibril level. Achilles tendons primarily contain longitudinal collagen fibrils that braid spatially into a dense rope‐like collagen meshwork and are encapsulated or wound tightly by the oblique collagen fibrils emanating from the epitenon region. The arrangement of the collagen fibrils provides the longitudinal fibrils with essential structural integrity and endows the tendon with the unique mechanical function for withstanding tensile stresses. A novel 3D microscopic method has been developed to examine the 3D collagen microstructure of tendons without tissue dehydrating and labelling. The study also provides new knowledge about the collagen microstructure in an Achilles tendon, which enables understanding of the function of the tissue. The knowledge may be important for applying surgical and tissue engineering techniques to tendon reconstruction.     

基于相位一致性最大响应方向的虹膜识别方法

为了进一步提高虹膜识别的准确性,提出了一种基于相位一致性最大响应方向的虹膜识别方法.该方法对人眼图像进行图像预处理,得到归一化的虹膜图像;然后,提取像素对于相位一致性的最大响应方向作为可区分性特征,并将最大响应方向的序号编码为简单易处理的二进制虹膜代码;最后,通过计算两个虹膜代码之间的加权汉明距离衡量二者的差异,并给出识别结果.实验结果表明,该方法的等错率为0.871 5%,正确识别率为99.851 8%.与经典的虹膜识别方法相比,该方法具有更高的识别准确性.     

Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two‐photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy‐to‐operate platform capable to perform two‐photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

大视场多像机视频测量系统的全局标定

针对大视场多像机视频测量系统的全局标定,提出并实现了一种基于近景摄影测量技术的高精度像机标定方法.首先,设计一种带有标志点的十字架形标定参考物,十字架正反两个表面粘贴有环形的编码标志点和圆形的非编码标志点.然后,在现场用工业摄影测量系统XJTUDP重建出双面标定十字架的精确结构尺寸数据;将标定十字架在公共视场范围内依次摆出多个姿态,并控制多像机同步拍摄各姿态的图像.最后,为大视场像机选择10参数非线性成像模型,对采集的图像组依次进行标定运算,标定出各像机的内参数和畸变参数,并全局定向出各个像机在统一坐标系下的外方位参数.实验结果表明,该标定方法的重投影误差小于0.05 pixel;利用标定结果测量高精度标准尺长度,相对误差小于1/4 000,可满足大视场多像机视频测量对精度和效率的要求.     

分块峰值点局部区域生长的星像提取

为了获得高精度、高更新率的抗噪声性能,对星敏感器星像提取环节进行了研究。首先,分析星图中星像灰度的分布特点,建立了判断某个像素是否与峰值像素归属同一星像的标准。然后,介绍了像元阵列分块方法和背景预测法。最后,结合星像的特点提出了以峰值点为种子点的区域生长准则。仿真实验结果表明,在不加噪声的情况下,提取出的星像与参考星图完全一致,用质心法得到的亚像素定位精度为0.028 2。在添加均值为20、标准差高达2.5的强高斯灰度噪声的情况下,提取率仍能达到86.11%,质心精度则下降到0.219 6pixel。均匀性很差,信噪比低于4.9dB的实拍星图实验结果也证明该方法有很强的星像提取能力和准确性,能够满足强噪声弱星像质心提取的强抗干扰能力的要求。     

Nonlinear bio-photonic crystal effects revealed with multimodal nonlinear microscopy

Highly optically active nonlinear bio‐photonic crystalline and semicrystalline structures in living cells were studied by a novel multimodal nonlinear microscopy. Numerous biological structures, including stacked membranes and aligned protein structures are highly organized on a nanoscale and have been found to exhibit strong optical activities through second‐harmonic generation (SHG) interactions, behaving similarly to man‐made nonlinear photonic crystals. The microscopic technology used in this study is based on a combination of different imaging modes including SHG, third‐harmonic generation, and multiphoton‐induced fluorescence. With no energy release during harmonic generation processes, the nonlinear‐photonic‐crystal‐like SHG activity is useful for investigating the dynamics of structure–function relationships at subcellular levels and is ideal for studying living cells, as minimal or no preparation is required.     

High-resolution cryo-scanning electron microscopy study of the macromolecular structure of fibronectin fibrils

High-resolution cryo-scanning electron microscopy was used to examine fibronectin fibrils formed in culture by human skin fibroblasts and in a cell-free system by denaturing soluble plasma fibronectin with guanidine. These studies indicate that the conformation of fibrils formed in culture and in a cell-free system appeared to be similar and that fibronectin fibrils have at least two distinct structural conformations. Fibronectin fibrils can be very straight structures with smooth surfaces or highly nodular structures. The average diameter of the nodules in these fibrils is 12 nm. Both conformations can be seen within an individual fibril indicating that they are not different types of fibronectin fibrils but rather different conformational states. Immunolabeling studies with a monoclonal antibody, IST-2, to the heparin II binding domain of fibronectin revealed that the epitope was buried in highly smooth fibrils, but it was readily exposed in nodular fibrils. We propose, therefore, that fibronectin fibrils are highly flexible structures and, depending on the conformation of the fibril, certain epitopes on the surface may be buried or exposed.     

Optimization of Picrosirius red staining protocol to determine collagen fiber orientations in vaginal and uterine cervical tissues by Mueller polarized microscopy

Polarized microscopy provides unique information on anisotropic samples. In its most complete implementation, namely Mueller microscopy, this technique is well suited for the visualization of fibrillar proteins orientations, with collagen in the first place. However, the intrinsic optical anisotropy of unstained tissues has to be enhanced by Picrosirius Red (PR) staining to enable Mueller measurements. In this work, we compared the orientation mapping provided by Mueller and second harmonic generation (SHG) microscopies on PR stained samples of vaginal and uterine cervix tissues. SHG is a multiphoton technique that is highly specific to fibrillar collagen, and was taken as the “gold standard” for its visualization. We showed that Mueller microscopy can be safely used to determine collagen orientation in PR stained cervical tissue. In contrast, in vaginal samples, Mueller microscopy revealed orientations not only of collagen but also of other anisotropic structures. Thus PR is not fully specific to collagen, which necessitates comparison to SHG microscopy in every type of tissue. In addition to this study of PR specificity, we determined the optimal values of the staining parameters. We found that staining times of 5 min, and sample thicknesses of 5 µm were sufficient in cervical and vaginal tissues. Microsc. Res. Tech. 78:723–730, 2015. © 2015 Wiley Periodicals, Inc.     

Quantification of the graphical details of collagen fibrils in transmission electron micrographs

A novel 2D image analysis technique is demonstrated. Using the digitized images of articular cartilage from transmission electron microscopy (TEM), this technique performs a localized 'vector' analysis at each region that is large enough to include several or tens of collagen fibrils but small enough to provide a fine resolution for the whole tissue. For each small and localized region, the morphology of the collagen fibrils can be characterized by three quantities essential to the nature of the tissue: the concentration of the fibrils, the overall orientation of the fibrils, and the anisotropy of the fibrils. This technique is capable of providing new insight to the existing technology by assigning quantitative attributes to the qualitative graphics. The assigned quantities are sensitive to the fine structure of the collagen matrix and meaningful in the architectural nature of the collagen matrix. These quantities could provide a critical linkage between the ultrastructure of the tissue and the macroscopic behaviours of the material. In addition, coarse-graining the microscopic resolution of EM without compromising the essential features of the tissue's structure provides a direct view of the tissue's morphology and permits direct correlations and comparisons among interdisciplinary techniques.     

SFS与特征点对应技术结合的形状测量法研究

以特征点对应技术为基础的摄影测量术有着广泛应用,但存在着测量数据稀疏的缺陷,对此提出了SFS（Shape from shading）与特征点技术相结合的方法,加密插值点,解决复杂表面形状测量问题。首先设计了用于测量的摄影系统,然后讨论了图像平面的剖分算法,最后给出SFS与特征点技术结合的有限单元形式,并设计了多重网格加速求解算法。算例表明,该算法效果令人满意。     

应用正则化影响函数扩散模型的星图噪声滤波

由于恒星星图的噪声滤波对保持星点的边缘细节要求较高,本文以塔基（Tukey）扩散模型与改善的PM（Perona-Malik）模型为基础,提出了一种基于正则化影响函数扩散模型的星图噪声滤波方法。该方法通过导数算子提取边界点集,利用图像中原始像素和噪声像素的空间分布特性对图像进行噪声滤波处理,并通过给定边界条件恢复图像边缘。由于避免了方差稳定（VS）变换,该方法可以直接处理高斯噪声。对普通图像和添加高斯噪声星图进行了仿真测试,并与普通扩散函数算法进行了比较。实验结果表明：提出的算法表现出了较好的噪声滤波能力,同时有效地保持了特征图像的边缘。相对于普通扩散函数算法其平均绝对误差降低了13.6%,峰值信噪比平均提高了6.1%。得到的数据显示,本方法的滤波能力优于普通的扩散函数方法,特别适用于星图的噪声滤波处理。     

Observation of the ultrastructure of anterior cruciate ligament graft by atomic force microscopy

This study presented the fibril ultrastructure of retrieved grafts from the reconstruction of anterior cruciate ligament (ACL) using atomic force microscopy (AFM). The tapping mode images of the AFM were taken from different areas of the longitudinally cut grafts. Regular arrangement of collagen fibrils was found in certain areas of the graft. In many areas, however, the fibrils were not well arranged in a single direction, with some smaller fibrils oriented vertically to larger parallel fibrils. The crossing and tangling of fibrils in ACL grafts was well represented in the three‐dimensional AFM image. This abnormality of graft ultrastructure might indicate the possible alteration of the mechanical environment after ACL reconstruction. This study demonstrated the suitability and importance of ultrastructure observation of ACL grafts by AFM. SCANNING 31: 19–23, 2009. © 2009 Wiley Periodicals, Inc.     

Polarization confocal microscopy and Congo Red fluorescence: a simple and rapid method to determine the mean cellulose fibril orientation in plants

The mean or net preferential orientation of cellulose fibrils in plant cell walls is detected with polarization confocal laser scanning microscopy using the fluorescence dichroism of Congo Red. Single cells, arrays of cells in a tissue, or the epidermis of whole organs can be assayed in vivo . Aerial parts require an extra pectinase treatment because of the cuticle, which is impermeable to aqueous solutions. Peeling off the epidermis can be an elegant alternative, especially for leaves. With this method the net preferential fibril orientation can be related to the symmetry axis of the cell in quantitative terms. Data issuing from this approach are useful in current research on plant biomechanics.     

反射法测量固体材料的非线性系数研究

基于准线性近似理论研究有限幅值波的反射基波和二次谐波声场特性,采用多元高斯声束将其表征为平面波解、衍射项和衰减项组合的形式,推导出超声反射法测量固体纵波非线性系数的计算模型。对经垂直入射自由界面的反射二次谐波进行模拟分析,结果显示,尽管入射二次谐波的反射波与新产生的二次谐波相位相反导致总的反射二次谐波减弱,但当试块厚度大于1/2倍瑞利距离时,探头接受到的反射二次谐波可用于固体材料纵波非线性系数的有效测量。设计并开展了6061铝的纵波非线性系数测量试验,结果表明当考虑反射、衍射修正和衰减补偿后,反射法测得的纵波非线性系数值与透射法的测量结果相近,但相比透射法,反射法具有试验要求低、限制条件少等优势,为固体材料纵波非线性系数测量的实际应用提供了一种有效方法。     

基于高光谱图像的改进SIFT特征提取与匹配

针对尺度不变特征变换(SIFT)算法所提取图像特征点数量少、误匹率高的问题,提出了一种基于高光谱图像的改进SIFT算法。首先,依据传统SIFT算法中高斯金字塔的构造思想,结合在不同波段下的高光谱图像具有相同宏观特征的特点,首次用高光谱图像作为原始算法中经高斯变换产生的图像,使得检测到的具有实际意义的特征点数量大幅增加;其次,传统SIFT算法以及大量的改进方法都只通过目标象元邻域范围内的像素信息来构造特征描述符,而忽略了像素点的位置信息,文中将目标象元的位置信息纳入了特征描述符,在特征描述符的匹配阶段,在利用邻域范围内的像素信息进行粗匹配之后,利用特征描述符中的位置信息进行精细匹配。仿真实验结果表明在限定最优值与次优值之比的情况下,采用高光谱图像构造高斯金字塔的方式能显著增加特征点的提取数量,更多地挖掘出图像中的极值点;在特征描述符中加入目标象元的位置信息作为特征点匹配第二阶段的判断依据,正确匹配数量达到原方法的59倍以上,极大提升了算法的匹配性能。