Three-dimensional computer reconstruction of large tissue volumes based on composing series of high-resolution confocal images by GlueMRC and LinkMRC software

Computer-based visualization of large tissue volumes with high resolution based on composing series of high-resolution confocal images is presented. GlueMRC and LinkMRC programs are introduced, implementing composition of overlapping series of optical sections captured by a confocal microscope, registration and subsequent composition of successive confocal stacks. Both programs are using an interactive approach in combination with automatic algorithms for image registration. Further, the method for obtaining surface renderings of microscopical structure under study is described. For this purpose, structure contours visible in the sections are interactively digitized using a Colon plug-in module running in Ellipse environment. Then the coordinates of the contours are processed by special modules in the graphic programming environment IRIS Explorer and the structure surface is rendered. The method is shown on the 3-D reconstruction of the capillary bed of human placental villi and chick embryonic gut and its vascular bed.     

Alignment with sub‐pixel accuracy for images of multi‐modality microscopes using automatic calibration

A biological specimen is often imaged with various imaging modalities, and it is crucial that such images are well aligned to best reveal physiological structures and functions of the specimen for in‐depth analyses. In this paper, we present a methodology for automatic calibration of multiple optical imaging modalities within the x–y detector plane using a custom chrome‐on‐glass target and an automatic and accurate registration algorithm. The target contains lines crossing at random angles, and our method of registration is based on the alignment of salient features extracted from the lines within the individual images. Once spatial relationships are found between the various detectors and applied to the resultant images, no further registration is required for all static samples, and the registered images serve as the starting point for registration of dynamic samples, where the remaining misalignment is caused by sample movement. We have validated our algorithm with 40 inter‐modal and 30 intra‐modal image pairs, and the success rates are 95 and 100%, respectively, with sub‐pixel accuracy. This methodology is widely applicable to any multi‐modal microscope that combines a number of imaging modalities on a common platform assuming images of the target can be obtained.     

Automatic image acquisition, calibration and montage assembly for biological X-ray microscopy

We describe a system for the automatic acquisition and processing of digital images in a high-resolution X-ray microscope, including the formation of large-field high-resolution image montages. A computer-controlled sample positioning stage provides approximate coordinates for each high-resolution subimage. Individual subimages are corrected to compensate for time-varying, non-uniform illumination and CCD-related artefacts. They are then automatically assembled into a montage. The montage assembly algorithm is designed to use the overlap between each subimage and multiple neighbours to improve the performance of the registration step and the fidelity of the result. This is accomplished by explicit use of recorded stage positions, optimized ordering of subimage insertion, and registration of subimages to the developing montage. Using this procedure registration errors are below the resolution limit of the microscope (43 nm). The image produced is a seamless, large-field montage at full resolution, assembled automatically without human intervention. Beyond this, it is also an accurate X-ray transmission map that allows the quantitative measurement of anatomical and chemical features of the sample. Applying these tools to a biological problem, we have conducted the largest X-ray microscopical study to date.     

Using the Hilbert transform for 3D visualization of differential interference contrast microscope images

Differential interference contrast (DIC) is frequently used in conventional 2D biological microscopy. Our recent investigations into producing a 3D DIC microscope (in both conventional and confocal modes) have uncovered a fundamental difficulty: namely that the phase gradient images of DIC microscopy cannot be visualized using standard digital image processing and reconstruction techniques, as commonly used elsewhere in microscopy. We discuss two approaches to the problem of preparing gradient images for 3D visualization: integration and the Hilbert transform. After applying the Hilbert transform, the dataset can then be visualized in 3D using standard techniques. We find that the Hilbert transform provides a rapid qualitative pre-processing technique for 3D visualization for a wide range of biological specimens in DIC microscopy, including chromosomes, which we use in this study.     

3D volumes constructed from pixel-based images by digitally clearing plant and animal tissue

Construction of three-dimensional volumes from a series of two-dimensional images has been restricted by the limited capacity to decrease the opacity of tissue. The use of commercial software that allows colour-keying and manipulation of two-dimensional images in true three-dimensional space allowed us to construct three-dimensional volumes from pixel-based images of stained plant and animal tissue without generating vector information. We present three-dimensional volumes of (1) the crown of an oat plant showing internal responses to a freezing treatment, (2) a sample of a hepatocellular carcinoma from a woodchuck liver that had been heat-treated with computer-guided radiofrequency ablation to induce necrosis in the central portion of the tumour, and (3) several features of a sample of mouse lung. The technique is well suited to images from large sections (greater than 1 mm) generated from paraffin-embedded tissues. It is widely applicable, having potential to recover three-dimensional information at virtually any resolution inherent in images generated by light microscopy, computer tomography, magnetic resonance imaging or electron microscopy.     

3‐D PSF fitting for fluorescence microscopy: implementation and localization application

Localization microscopy relies on computationally efficient Gaussian approximations of the point spread function for the calculation of fluorophore positions. Theoretical predictions show that under specific experimental conditions, localization accuracy is significantly improved when the localization is performed using a more realistic model. Here, we show how this can be achieved by considering three‐dimensional (3‐D) point spread function models for the wide field microscope. We introduce a least‐squares point spread function fitting framework that utilizes the Gibson and Lanni model and propose a computationally efficient way for evaluating its derivative functions. We demonstrate the usefulness of the proposed approach with algorithms for particle localization and defocus estimation, both implemented as plugins for ImageJ.     

Confocal bi-protocol: a new strategy for isotropic 3D live cell imaging

The conventional approach for microscopic 3D cellular imaging is based on axial through-stack image series which has some significant limitations such as anisotropic resolution and axial aberration. To overcome these drawbacks, we have recently introduced an alternative approach based on micro-rotation image series. Unfortunately, this new technique suffers from a huge burden of computation that makes its use quite difficult for current applications. To address these problems we propose a new imaging strategy called bi-protocol, which consists of coupling micro-rotation acquisition and conventional z-stack acquisition. We experimentally prove bi-protocol 3D reconstruction produces similar quality to that of pure micro-rotation, but offers the advantage of reduced computation burden because it uses the z-stack volume to accelerate the registration of the micro-rotation images.     

基于激光扫描和SFM的非同步点云三维重构方法

室外场景具有测量数据量大、扫描数据易重叠及建筑物表面信息复杂等特点,单靠激光扫描方法能够获得场景精确的深度信息,但缺乏颜色和纹理信息,利用从运动中恢复结构(SFM)方法可获得丰富的彩色信息,但重构精度不高,若将两种设备固定进行在线实时同步测量,易受到测量环境和系统制约不易实现。针对此问题,提出了一种基于激光扫描和SFM结合的非同步点云数据融合的三维重构方法。首先,提出利用手动选择控制点进行7自由度初始配准,再利用迭代最近点(ICP)算法对初始配准结果进行精确配准,最后利用最近点搜索算法将分布在经基于面片的多视图立体视觉(PMVS)算法优化后的SFM数据中的颜色信息与激光扫描的点云坐标进行融合。实验结果和数据分析显示,本文的方法能有效地将激光扫描与SFM点云数据进行融合,实现了室外大场景的三维彩色重构。     

XuvTools: free, fast and reliable stitching of large 3D datasets

Current biomedical research increasingly requires imaging large and thick 3D structures at high resolution. Prominent examples are the tracking of fine filaments over long distances in brain slices, or the localization of gene expression or cell migration in whole animals like Caenorhabditis elegans or zebrafish. To obtain both high resolution and a large field of view (FOV), a combination of multiple recordings (‘tiles’) is one of the options. Although hardware solutions exist for fast and reproducible acquisition of multiple 3D tiles, generic software solutions are missing to assemble (‘stitch’) these tiles quickly and accurately. In this paper, we present a framework that achieves fully automated recombination of tiles recorded at arbitrary positions in 3D space, as long as some small overlap between tiles is provided. A fully automated 3D correlation between all tiles is achieved such that no manual interaction or prior knowledge about tile positions is needed. We use (1) phase‐only correlation in a multi‐scale approach to estimate the coarse positions, (2) normalized cross‐correlation of small patches extracted at salient points to obtain the precise matches, (3) find the globally optimal placement for all tiles by a singular value decomposition and (4) accomplish a nearly seamless stitching by a bleaching correction at the tile borders. If the dataset contains multiple channels, all channels are used to obtain the best matches between tiles. For speedup we employ a heuristic method to prune unneeded correlations, and compute all correlations via the fast Fourier transform (FFT), thereby achieving very good runtime performance. We demonstrate the successful application of the proposed framework to a wide range of different datasets from whole zebrafish embryos and C. elegans, mouse and rat brain slices and fine plant hairs (trichome). Further, we compare our stitching results to those of other commercially and freely available software solutions. The algorithms presented are being made available freely as an open source toolset ‘XuvTools’ at the corresponding author's website ( http://lmb.informatik.uni‐freiburg.de/people/ronneber ), licensed under the GNU General Public License (GPL) v2. Binaries are provided for Linux and Microsoft Windows. The toolset is written in templated C++, such that it can operate on datasets with any bit‐depth. Due to the consequent use of 64bit addressing, stacks of arbitrary size (i.e. larger than 4 GB) can be stitched. The runtime on a standard desktop computer is in the range of a few minutes. A user friendly interface for advanced manual interaction and visualization is also available.     

6-3-3并联机构尺度的优化综合

提出了以满足6-3-3并联机构使用功能为准则的尺度综合优化建模方法,即以功能要求的任务轨迹为动平台运动规划路径,以尺度参数和任务轨迹在工作空间的定位参数为设计变量,以机构空间结构尽可能紧凑,而工作空间尽可能大为目标函数、工作空间涵盖任务轨迹、主动件移动范围和关节转角不超过设定极限值为约束,建立优化模型并通过软件编程实现该模型的求解。最后,给出了一个算例。     

An open‐source deconvolution software package for 3‐D quantitative fluorescence microscopy imaging

Deconvolution techniques have been widely used for restoring the 3‐D quantitative information of an unknown specimen observed using a wide‐field fluorescence microscope. Deconv , an open‐source deconvolution software package, was developed for 3‐D quantitative fluorescence microscopy imaging and was released under the GNU Public License. Deconv provides numerical routines for simulation of a 3‐D point spread function and deconvolution routines implemented three constrained iterative deconvolution algorithms: one based on a Poisson noise model and two others based on a Gaussian noise model. These algorithms are presented and evaluated using synthetic images and experimentally obtained microscope images, and the use of the library is explained. Deconv allows users to assess the utility of these deconvolution algorithms and to determine which are suited for a particular imaging application. The design of Deconv makes it easy for deconvolution capabilities to be incorporated into existing imaging applications.     

汽车差速器结构设计、三维建模与虚拟装配研究

简述了依据汽车设计规范对差速器各零件进行结构设计的主要思路,给出了在Pro／E软件中基于特征创建差速器各零件三维模型的思路与结果,阐述了建立差速器三维装配模型的方法和主要步骤。差速器各零件三维精确建模有助于提高其零件的数控加工精度,三维虚拟装配则有助于及时发现和解决结构设计中的问题,从而缩短差速器产品的研发周期,降低设计成本。     

Dynamics of subcellular organelles, growth hormone, Rab3B, SNAP-25, and syntaxin in rat pituitary cells caused by growth hormone releasing hormone and somatostatin

Rab3B is involved in the exocytosis of synaptic vesicles and secretory granules in the central nervous system and the anterior pituitary cells. The aim of this study was to elucidate both the role of rab3B in GH secretion and the mutual relationship of rab3B and SNARE proteins. Adult male rats were injected intravenously with 10 microg of growth hormone releasing hormone (GHRH) or 10 microg of somatostatin (SRIF). Untreated rats were used as controls, and their pituitary glands were sectioned for histochemical examination. Rab3B is localized on the limiting membrane of the secretory granules and the cytosol. Confocal laser scanning microscopic observation of immunohistochemical double staining of rab3B and GH revealed that immunoreactivity of rab3B increased in GHRH-treated rats and decreased in SRIF-treated rats. Confocal laser scanning microscopic observation of immunohistochemical double staining of SNAP-25, syntaxin, and rab3B revealed the co-localization of rab3B and these SNARE proteins in GHRH-treated rats, and their dissociation in SRIF-treated rats. These results suggest that rab3B plays a principal role in GH secretion in the anterior pituitary cells and that SNAP-25 and syntaxin act as co-workers with rab3B in the functional regulation of GH secretion.     

Quantitative fractography by digital image processing: NIH Image macro tools for stereo pair analysis and 3-D reconstruction

A set of NIH Image macro programs was developed to make qualitative and quantitative analyses from digital stereo pictures produced by scanning electron microscopes. These tools were designed for image alignment, anaglyph representation, animation, reconstruction of true elevation surfaces, reconstruction of elevation profiles, true-scale elevation mapping and, for the quantitative approach, surface area and roughness calculations. Limitations on time processing, scanning techniques and programming concepts are also discussed.     

Multiscale iterative voting for differential analysis of stress response for 2D and 3D cell culture models

Focused ion beam-scanning electron microscope (FIB-SEM) tomography is a powerful application in obtaining three-dimensional (3D) information. The FIB creates a cross section and subsequently removes thin slices. The SEM takes images using secondary or backscattered electrons, or maps every slice using X-rays and/or electron backscatter diffraction patterns. The objective of this study is to assess the possibilities of combining FIB-SEM tomography with cathodoluminescence (CL) imaging. The intensity of CL emission is related to variations in defect or impurity concentrations. A potential problem with FIB-SEM CL tomography is that ion milling may change the defect state of the material and the CL emission. In addition the conventional tilted sample geometry used in FIB-SEM tomography is not compatible with conventional CL detectors. Here we examine the influence of the FIB on CL emission in natural diamond and the feasibility of FIB-SEM CL tomography. A systematic investigation establishes that the ion beam influences CL emission of diamond, with a dependency on both the ion beam and electron beam acceleration voltage. CL emission in natural diamond is enhanced particularly at low ion beam and electron beam voltages. This enhancement of the CL emission can be partly explained by an increase in surface defects induced by ion milling. CL emission enhancement could be used to improve the CL image quality. To conduct FIB-SEM CL tomography, a recently developed novel specimen geometry is adopted to enable sequential ion milling and CL imaging on an untilted sample. We show that CL imaging can be manually combined with FIB-SEM tomography with a modified protocol for 3D microstructure reconstruction. In principle, automated FIB-SEM CL tomography should be feasible, provided that dedicated CL detectors are developed that allow subsequent milling and CL imaging without manual intervention, as the current CL detector needs to be manually retracted before a slice can be milled. Due to the required high electron beam acceleration voltage for CL emission, the resolution for FIB-SEM CL tomography is currently limited to several hundreds of nm in XY and up to 650 nm in Z for diamonds. Opaque materials are likely to have an improved Z resolution, as CL emission generated deeper in the material is not able to escape from it.     

Stereo vision for 3D measurement: accuracy analysis, calibration and industrial applications

This paper evaluates the accuracy of different camera calibration and measurement methods used in 3D stereo vision with CCD cameras. These methods are evaluated by means of several precision tests, determining their error limits under specified conditions of operation. To check the precision of such systems, a CMM and some calibration objects, such as grids, plates, spheres, etc. are used. Two practical applications are described: a cost-effective system for the measurement of free-form surfaces, able to generate CAD models and measuring programs for CMMs. The system aims to reduce some difficulties associated with stereo vision and to speed up the traditional digitizing process. The other application involves car frame measurement. A new automatic measuring system has been developed, allowing contactless car frame measurement through two rotating CCD cameras.     

Rapid overlapping-volume acquisition and reconstruction (ROVAR): automated 3D tiling for high-resolution, large field-of-view optical microscopy

Micrometer-scale three-dimensional data from fluorescence microscopes offer unique insight into cellular morphology and function by resolving subcellular locations of fluorescent dyes and proteins. To increase field-of-view size while using a high-resolution multiphoton microscope, we have created an automated system of rapidly acquiring overlapping image stacks from multiple fields-of-view along a nonplanar tissue surface. Each image stack is acquired only between the surface and the maximal penetrating depth, as determined by the image signal-to-background ratio. This results in the acquisition of the volume containing visible tissue along the tissue surface, excluding the empty volume above the tissue and the volume beyond the maximum imaging depth within the tissue. The automated collection of overlapping volumes is followed by reconstruction that can efficiently generate a single three-dimensional volume of the tissue surface. This approach yields data spanning multiple millimetres at micrometre resolution that is faster while requiring less work from the microscope operator. The advantages of the system are demonstrated by acquisition of data from intact, unfixed organs without a coverglass both in vivo and in situ.     

KSpaceNavigator as a tool for computer-assisted sample tilting in high-resolution imaging, tomography and defect analysis

This article describes a novel software tool, the KSpaceNavigator, which combines sample stage and crystallographic coordinates in a control sphere. It also provides simulated kinematic diffraction spot patterns, Kikuchi line patterns and a unit cell view in real time, thus allowing intuitive and transparent navigation in reciprocal space. By the overlay of experimental data with the simulations and some interactive alignment algorithms, zone axis orientations of the sample can be accessed quickly and with great ease. The software can be configured to work with any double-tilt or tilt–rotation stage and overcomes nonlinearities in existing goniometers by lookup tables. A subroutine for matching the polyhedral shape of a nanoparticle assists with 3D analysis and modeling. The new possibilities are demonstrated with the case of a faceted BaTiO₃ nanoparticle, which is tilted into three low-index zone axes using the piezo-controlled TEAM stage, and with a multiply twinned tetrahedral Ge precipitate in Al, which is tilted into four equivalent zone axes using a conventional double-tilt stage. Applications to other experimental scenarios are also outlined.     

The saucor, a new stereological tool for analysing the spatial distributions of cells, exemplified by human neocortical neurons and glial cells

The 3D spatial arrangement of particles or cells, for example glial cells, with respect to other particles or cells, for example neurons, can be characterized by the radial number density function, which expresses the number density of so-called 'secondary' particles as a function of their distance to a 'primary' particle. The present paper introduces a new stereological method, the saucor, for estimating the radial number density using thick isotropic uniform random or vertical uniform random sections. In the first estimation step, primary particles are registered in a disector. Subsequently, smaller counting windows are drawn with random orientation around every primary particle, and the positions of all secondary particles within the windows are recorded. The shape of the counting windows is designed such that a large portion of the volume close to the primary particle is examined and a smaller portion of the volume as the distance to the primary object increases. The experimenter can determine the relation between these volumina as a function of the distance by adjusting the parameters of the window graph, and thus reach a good balance between workload and obtained information. Estimation formulae based on the Horvitz-Thompson theorem are derived for both isotropic uniform random and vertical uniform random designs. The method is illustrated with an example where the radial number density of neurons and glial cells around neurons in the human neocortex is estimated using thick vertical sections for light microscopy. The results indicate that the glial cells are clustered around the neurons and the neurons have a tendency towards repulsion from each other.