Automated quantification and reconstruction of collagen matrix from 3D confocal datasets

The geometrical structure of fibrous extracellular matrix (ECM) impacts on its biological function. In this report, we demonstrate a new algorithm designed to extract quantitative structural information about individual collagen fibres (orientation, length and diameter) from 3D backscattered‐light confocal images of collagen gels. The computed quantitative data allowed us to create surface‐rendered 3D images of the investigated sample.     

MicroCT examination of human bone specimens: effects of polymethylmethacrylate embedding on structural parameters

X‐ray microtomography permits the nondestructive investigation of trabecular and cortical bone specimens without special preparation of the sample. To do a quantitative characterization, the cross‐section images have to be binarized, separating bone from nonbone. For this purpose, a widely used method is uniform thresholding. However, for commonly available microtomography scanners which use a polychromatic X‐ray source, it is unclear what effect the surrounding medium (e.g. air, saline solution, polymethylmethacrylate) has on the threshold value used for the binarization. In the literature an easy procedure to find the optimal uniform threshold value for a given acquisition condition is reported. By applying this procedure, the present work investigated whether a microtomography scan of trabecular bone samples in air or embedded in polymethylmethacrylate gave the same results in terms of structural parameters. The gold standard, that is, histological sections, was used as a reference. Two fixed threshold values were found, one for the microtomography scans performed in air and one for the scans with the same samples embedded in polymethylmethacrylate. These were applied on the correspondent microtomography images for the estimation of structural parameters, such as bone volume fraction, direct trabecular thickness, direct trabecular separation and structure model index. Paired comparisons were made in bone volume fraction between histological sections and microtomography cross‐sections for the same bone samples scanned first in air and then embedded in polymethylmethacrylate, by which no significant differences were found. Paired comparisons were also made in bone volume fraction, direct trabecular thickness, direct trabecular separation and structure model index for the same samples over volumes of interest of 4 × 4 × 4 mm³ between microtomography scans in air and scans with the samples embedded in polymethylmethacrylate. Neither these comparisons showed significant differences. This leads to the conclusion that structural parameters estimated by microtomography for human trabecular bone samples scanned either in air or embedded in polymethylmethacrylate are not affected by the surrounding medium (i.e. presence or absence of polymethylmethacrylate), provided that the corresponding optimal threshold value is applied for each acquisition condition.     

Development of preparation methods for and insights obtained from atomic force microscopy of fluid spaces in cortical bone

Several preparation methods were developed to investigate the dimensions and surface structure of fluid spaces within cortical bone, using atomic force microscopy (AFM). Of special interest was the morphology of the lacunocanalicular system, which serves as a conduit between osteocytes encased in bone tissue, the intramedullary cavity, blood vessels running through the bone, and the periosteal surface of bone. Fracture and the removal of either the mineral or the organic component is a method by which each component can be investigated at a very high resolution in situ. Although fractured bone was too rough to image details of the lacunocanalicular system, post-treatment with ethylene diamine tetraacetic acid (EDTA) or papain allowed for investigation of the collagen matrix or the mineral crystals of bone, respectively. Cut and polished bone was smooth enough for identifying the lacunae of bone using AFM, but unambiguous differentiation between the canaliculi and cracks in the bone surface was not possible. However, when the lacunocanalicular system was filled with polymethylmethacrylate (PMMA), it was possible to image casts of the lacunocanalicular system by selectively etching away the surrounding bone matrix. Using this method, we identified individual canaliculi and measured their dimensions. Furthermore, by carefully etching away the bone matrix in successive etches, it was shown that the wall structure of the canaliculus is dominated by collagen fibrils. These observations have important implications for fluid flow in bone.     

An investigation of segmentation methods and texture analysis applied to tomographic images of human vertebral cancellous bone

The goal of this study is to determine architectural and textural parameters on computed tomographic (CT) images, allowing us to explain the mechanical compressive properties of bone. Although the resolution (150 μm) is of the same order of magnitude as the trabecular thickness, this method enables the possibility of perfecting an in vivo peripheral CT system with an acceptable radiation dose for the patient. This study was performed on L2 vertebrae cancellous bone specimens taken after necropsy in 22 subjects aged 47–95 years (mean: 79 years). The segmentation process is a crucial point in the determination of accurate architectural parameters. In this paper the use of two different segmentation methods is investigated, based on an edge enhancement and a region growing approach. The images are compared and the architectural parameters extracted from the images segmented by both methods lead to a quantitative evaluation. The parameters are found to be globally robust towards the segmentation process, although some of them are much more sensitive to the approach used. Highly significant correlations ( P  < 0.0005) have been obtained between the two segmentation methods for all the parameters, with ρ ranging from 0.70 to 0.93. In order to improve the assessment of bone architecture, texture analysis (run length method) was investigated. New features are obtained from an image reduced to 16 grey-levels. Textural parameters in addition to architectural parameters in a multivariate regression model increase significantly ( P  = 0.01) the prediction of the maximum compressive strength (variation of r ² from 0.75 up to 0.89).     

Quantitative morphological analysis of curvilinear network for microscopic image based on individual fibre segmentation (IFS)

Microscopic images of curvilinear fibre network structure like cytoskeleton are traditionally analysed by qualitative observation, which can hardly provide quantitative information of their morphological properties. However, such information is crucially contributive to the understanding of important biological events, even helps to learn about the inner relations hard to perceive. Individual fibre segmentation–based curvilinear structure detector proposed in this study can identify each individual fibre in the network, as well as connections between different fibres. Quantitative information of each individual fibre, including length, orientation and position, can be extracted; so are the connecting modes in the fibre network, such as bifurcation, intersection and overlap. Distribution of fibres with different morphological properties is also presented. No manual intervening or subjective judging is required in the analysing process. Both synthesized and experimental microscopic images have verified that the detector is capable to segment curvilinear network at the subcellular level with strong noise immunity. The proposed detector is finally applied to the morphological study on cytoskeleton. It is believed that the individual fibre segmentation–based curvilinear structure detector can greatly enhance our understanding of those biological images generated from tons of biological experiments.     

Stereoscopic display of atomic force microscope images using anaglyph techniques

This paper describes the use of a standard stereo-pair image display method for presenting the three-dimensional relief information found in atomic force microscope (AFM) images. The method makes use of commercially available image processing software packages. The techniques are illustrated on AFM images of the cuticle structure of a human hair fibre.     

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.     

Accurate marker-free alignment with simultaneous geometry determination and reconstruction of tilt series in electron tomography

An image alignment method for electron tomography is presented which is based on cross-correlation techniques and which includes a simultaneous refinement of the tilt geometry. A coarsely aligned tilt series is iteratively refined with a procedure consisting of two steps for each cycle: area matching and subsequent geometry correction. The first step, area matching, brings into register equivalent specimen regions in all images of the tilt series. It determines four parameters of a linear two-dimensional transformation, not just translation and rotation as is done during the preceding coarse alignment with conventional methods. The refinement procedure also differs from earlier methods in that the alignment references are now computed from already aligned images by reprojection of a backprojected volume. The second step, geometry correction, refines the initially inaccurate estimates of the geometrical parameters, including the direction of the tilt axis, a tilt angle offset, and the inclination of the specimen with respect to the support film or specimen holder. The correction values serve as an indicator for the progress of the refinement. For each new iteration, the correction values are used to compute an updated set of geometry parameters by a least squares fit. Model calculations show that it is essential to refine the geometrical parameters as well as the accurate alignment of the images to obtain a faithful map of the original structure.     

Analysis of AFM Images of Self-Structured Surface Textures by Directional Fractal Signature Method

A new method, called augmented blanket with rotating grid (ABRG), has been proposed in our recent work on characterizing roughness and directionality of self-structured surface textures. This is the first method that calculates fractal dimensions (FDs) at individual scales and directions for the entire surface image data and does not require the data to be Brownian fractal. However, before the ABRG method can be used in real applications, effects of atomic force microscope (AFM) imaging conditions on FDs need to be evaluated first. In this paper, computer-generated AFM images with three different resolutions, 48 combinations of tip radii and cone angles, and 15 noise levels were used in the tests. The images represent isotropic self-structured surface textures with small, medium and large motif sizes, and anisotropic surfaces exhibiting two dominating directions. For isotropic surfaces, the ABRG method is not significantly affected (i.e. FDs changes <5 %) by image resolution, tip size (for surfaces with large motifs) and noise (except the level above 8 %). For anisotropic surfaces, the method exhibits large changes in FDs (up to ?34 %). The results obtained show that the ABRG method can be effective in analysing the AFM images of self-structured surface textures. However, some precautions should be taken with anisotropic and isotropic surfaces with small motifs.     

原子力显微镜(AFM)在光盘检测及其质量控制中的应用

综述了原子力显微镜(AFM)在光盘质量检测中的应用.AFM能够在nm尺度上直接对光盘及其模板上的信息位几何结构的特征尺寸及其误差进行三维测量,从而可以建立生产工艺参数和信息位几何结构之间、信息位几何结构和盘片电气性能之间的关系,进而找出影响光盘质量的直接原因.用AFM进行光盘质量检测主要有三方面：盘片和模板表面的定性观测;信息位几何结构的半定量分析;信息位特征尺寸的统计分析.定性观测和半定量分析可以对盘片播放的高误差率、凹坑形态和块出错率、凸台形态及其表面粗糙度等参数进行有针对性的检测;而信息位特征尺寸的统计分析则可以对信息位几何结构的关键参数进行面向生产过程的统计分析.所得结论表明AFM在光盘质量检测过程中具有独特的优势.     

Abrasive flow machining applied to plastic gear matrix polishing

The topic of this paper is the application of abrasive flow machining (AFM) to gear tool inserts polishing. Polished surface on plastic gear teeth improves surface geometry stability, and it increases the lifespan, which was proved on the gear testing rig. Experiments have shown that it is an efficient alternative to the hand polishing procedure. Besides significant cost and processing time savings, AFM generates constant surface quality. The achieved roughness is homogeneous on the entire machined surface; it is reduced from R _a?=?0.68 μm to R _a?=?0.08 μm in 120 s. At the same time, the tooth geometry profile is not damaged. The first time, surface polishing should be done at request because of individually manufactured tool inserts. Processing parameters depend on the type of the abrasive machine, the polishing paste and part geometry. Computer-aided abrasive flow analyses and practical experiments assist in setting optimum AFM process parameters. The paper presents a working set of parameters and a detailed report on machined surface measurement data. On the base of better understanding of AFM process, the surface roughness prediction model and thickness of removed material model was setup. It has high accuracy and reliability for specific applications. The use of plastic gears for various applications is widespread; the presented process improvement is an important innovation for injection molding tools manufacturers.     

A simple method for AFM tip characterization by polystyrene spheres

An AFM image would not be the true topography of a surface because of the limitation of a finite size of the tip. The true topography of the surface can be deduced if we can know the tip shape. In this paper a simple method has been established to determine the profile of an AFM tip. A geometrical model for the tip and a spherical object has been proposed to show the procedure for deducing the tip shape from AFM images. Isolated spheres and closely packed spheres with different diameters have been observed to confirm the tip shape by this method. It is a non-destructive method to determine the tip shape and the results can be used for future reconstruction of an AFM image.     

Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples

A new device (NTEGRA Tomo) that is based on the integration of the scanning probe microscope (SPM) (NT‐MDT NTEGRA SPM) and the Ultramicrotome (Leica UC6NT) is presented. This integration enables the direct monitoring of a block face surface immediately following each sectioning cycle of ultramicrotome sectioning procedure. Consequently, this device can be applied for a serial section tomography of the wide range of biological and polymer materials. The automation of the sectioning/scanning cycle allows one to acquire up to 10 consecutive sectioned layer images per hour. It also permits to build a 3‐D nanotomography image reconstructed from several tens of layer images within one measurement session. The thickness of the layers can be varied from 20 to 2000 nm, and can be controlled directly by its interference colour in water. Additionally, the NTEGRA Tomo with its nanometer resolution is a valid instrument narrowing and highlighting an area of special interest within volume of the sample. For embedded biological objects the ultimate resolution of SPM mostly depends on the quality of macromolecular preservation of the biomaterial during sample preparation procedure. For most polymer materials it is comparable to transmission electron microscopy (TEM). The NTEGRA Tomo can routinely collect complementary AFM and TEM images. The block face of biological or polymer sample is investigated by AFM, whereas the last ultrathin section is analyzed with TEM after a staining procedure. Using the combination of both of these ultrastructural methods for the analysis of the same particular organelle or polymer constituent leads to a breakthrough in AFM/TEM image interpretation. Finally, new complementary aspects of the object's ultrastructure can be revealed.     

Improved algorithms for automatic bone histomorphometry on a numerized image analysis system

Quantitative histological methods have proved to be the most effective methods in bone disease research. Faster and more accurate techniques are currently needed. We have developed a simple digitized image analysis system which allows accurate measurements of trabecular bone mass. The algorithm is based on the 'four-connected sets' mathematical theory. Given a numerized image displayed by a CCD camera, the algorithm recognizes all possible four-connected sets and provides area measurements. The first procedure automatically eliminates small, irrelevant profiles (wrinkles, cell nuclei, etc.) while larger profiles are erased interactively. The second procedure similarly erases the artefactual defects within the trabecule (artefactual cracks or empty osteocytic lacunae). The method was shown to be very accurate and time-saving.     

Carbon nanotubes: a promising standard for quantitative evaluation of AFM tip apex geometry

The direct contact between tip and sample in atomic force microscopy (AFM) leads to demand for a quantitative knowledge of the AFM tip apex geometry in high-resolution AFM imaging and many other types of AFM applications like force measurements and surface roughness measurements. Given, the AFM tip apex may change continuously during measurements due to wear or during storage due to oxidation, it is very desirable to develop an easy and quick way for quantitative evaluation of AFM tip radius when necessary. In this study, we present an efficient method based on Zenhausern model (Scanning 14 (1992) 212) by measuring single-wall carbon nanotubes deposited on a flat substrate to reach this goal. Experimental results show the method can be used for routine quantitative evaluation of AFM tip apex geometry for tips with effective radii down to the nanometer scale.     

Polystyrene spheres on mica substrates: AFM calibration, tip parameters and scan artefacts

Atomic force microscopy (AFM), in various versions, has had major impact as a surface structural and spectroscopic tool since its invention in 1986. At its present state of development, however, the interpretation of AFM images is limited by the current state of methodologies for calibration over the wide dynamic range of magnification. Also, the parameters of individual tips, as well as the generic characteristics of different kinds of tips, affect both the quality of the images and their interpretation. Finally, the very nature of the tip-to-surface interaction will generate artefacts, in addition to those associated with tip shape, which need to be fully understood by the practitioners of force microscopy. This project seeks to address and shed light on some of these issues. Polystyrene beads deposited on mica substrates form hexagonal close-packed layers. The unit cell parameters are suitable for calibration of the AFM in the lateral plane, while the perpendicular spacing of the layers is appropriate for calibration along the vertical axis. Using different size fractions, it is straightforward to determine the extents of linearity, orthogonality, thermal and instrumental drifts over distances from 100 nm to tens of micrometres. The present results show that the methodologies for contact mode operation can be adapted to noncontact modes. It is known that an AFM image arises from a convolution of surface topography and tip shape, and is mediated by the interaction. In principle it is possible to carry out a deconvolution, if we have complete knowledge about two of the three elements (i.e. tip, surface and interaction). In practice we rarely have the requisite information. Prominent artefacts will occur when the characteristic parameters of the tip are comparable to those of the surface topography, and/or if there is a variable strength, or extent of localization, of the interaction. The present results demonstrate artefacts due to effects of geometry as well as interaction.     

一维矩形光栅AFM图像盲探针表面重建模拟研究

探针结构参数的合理选取将直接决定扫描图像及其盲探针修正图像的失真程度。基于此,以一维矩形模拟光栅为典型案例,对该模拟光栅的原子力显微镜(AFM)扫描成像过程与盲探针修正过程进行了仿真,阐明了探针结构参数对扫描成像过程与盲探针修正过程的影响规律。通过建立线宽变化度与半高宽相结合的图像重建误差评价指标,确定了针对该模拟光栅的AFM探针建议结构参数,并取得了良好的光栅图像重建效果。研究表明,应用线宽变化度结合半高宽来综合评价光栅的AFM测量和图像重建过程,有利于提升实际光栅AFM图像盲探针重建的准确度。     

Sensitivity of proximal femoral stiffness and areal bone mineral density to changes in bone geometry and density

Areal bone mineral density (aBMD) is the most common surrogate measurement for assessing the bone strength of the proximal femur associated with osteoporosis. Additional factors, however, contribute to the overall strength of the proximal femur, primarily the anatomical geometry. Finite element analysis (FEA) is an effective and widely used computer-based simulation technique for modelling mechanical loading of various engineering structures, providing predictions of displacement and induced stress distribution due to the applied load. FEA is therefore inherently dependent upon both density and anatomical geometry. FEA may be performed on both three-dimensional and two-dimensional models of the proximal femur derived from radiographic images, from which the mechanical stiffness may be predicted. It is examined whether the outcome measures of two-dimensional FEA, two-dimensional, finite element analysis of X-ray images (FEXI), and three-dimensional FEA computed stiffness values of the proximal femur are more sensitive than aBMD to changes in trabecular bone density and femur geometry. It is assumed that if an outcome measure follows known trends with changes in density and geometric parameters, then an increased sensitivity will be indicative of an improved prediction of bone strength. All three outcome measures increased non-linearly with trabecular bone density, increased linearly with cortical shell thickness and neck width, decreased linearly with neck length, and were relatively insensitive to neck-shaft angle. For femoral head radius, aBMD was relatively insensitive, with two-dimensional FEXI and three-dimensional FEA demonstrating a non-linear increase and decrease in sensitivity respectively. For neck anteversion, aBMD decreased non-linearly, whereas both two-dimensional FEXI and three-dimensional FEA demonstrated a parabolic-type relationship, with the maximum stiffness being achieved at an angle of approximately 15 degrees. Multi-parameter analysis showed that all three outcome measures demonstrated their highest sensitivity to a change in cortical thickness. When changes in all input parameters were considered simultaneously, three and two-dimensional FEA had statistically equal sensitivities (0.41 +/- 0.20 and 0.42 +/- 0.16 respectively, p = ns) that were significantly higher than the sensitivity of aBMD (0.24 +/- 0.07; p = 0.014 and 0.002 for three-dimensional and two-dimensional FEA respectively). This simulation study suggests that since mechanical integrity and FEA are inherently dependent on anatomical geometry, FEXI stiffness, being derived from conventional two-dimensional radiographic images, may provide an improvement in the prediction of bone strength of the proximal femur than currently provided by aBMD.     

Three-dimensional surface texture visualization of bone tissue through epifluorescence-based serial block face imaging

Serial block face imaging is a microscopy technique in which the top of a specimen is cut or ground away and a mosaic of images is collected of the newly revealed cross-section. Images collected from each slice are then digitally stacked to achieve 3D images. The development of fully automated image acquisition devices has made serial block face imaging more attractive by greatly reducing labour requirements. The technique is particularly attractive for studies of biological activity within cancellous bone as it has the capability of achieving direct, automated measures of biological and morphological traits and their associations with one another. When used with fluorescence microscopy, serial block face imaging has the potential to achieve 3D images of tissue as well as fluorescent markers of biological activity. Epifluorescence-based serial block face imaging presents a number of unique challenges for visualizing bone specimens due to noise generated by sub-surface signal and local variations in tissue autofluorescence. Here we present techniques for processing serial block face images of trabecular bone using a combination of non-uniform illumination correction, precise tiling of the mosaic in each cross-section, cross-section alignment for vertical stacking, removal of sub-surface signal and segmentation. The resulting techniques allow examination of bone surface texture that will enable 3D quantitative measures of biological processes in cancellous bone biopsies.