Synchrotron X-ray microscopic computed tomography of the pump system of a female mosquito

The pumping organ of blood-sucking female mosquitoes has a three-dimensional (3D) structure. However, conventional two-dimensional imaging methods are insufficient for visualizing the 3D structure in detail. Furthermore, their 3D imaging tasks are highly time consuming and sample preparation process requires elaborate skill. Among 3D imaging techniques, synchrotron X-ray microscopic computed tomography (SR-μCT) is especially suitable for small insects with opaque cuticles, such as mosquitoes. In this study, the 3D morphological structure of the pump system of a female mosquito was visualized using SR-μCT. Expandable volume capacities of two pump chambers were measured for several mosquito samples of similar size. To verify the cross-sectional images acquired by SR-μCT, complementary paraffin-sectioning data were compared.     

Automated microscopy system for mosaic acquisition and processing

An automatic mosaic acquisition and processing system for a multiphoton microscope is described for imaging large expanses of biological specimens at or near the resolution limit of light microscopy. In a mosaic, a larger image is created from a series of smaller images individually acquired systematically across a specimen. Mosaics allow wide‐field views of biological specimens to be acquired without sacrificing resolution, providing detailed views of biological specimens within context. The system is composed of a fast‐scanning, multiphoton, confocal microscope fitted with a motorized, high‐precision stage and custom‐developed software programs for automatic image acquisition, image normalization, image alignment and stitching. Our current capabilities allow us to acquire data sets comprised of thousands to tens of thousands of individual images per mosaic. The large number of individual images involved in creating a single mosaic necessitated software development to automate both the mosaic acquisition and processing steps. In this report, we describe the methods and challenges involved in the routine creation of very large scale mosaics from brain tissue labelled with multiple fluorescent probes.     

Atom probe trajectory mapping using experimental tip shape measurements

Atom probe tomography is an accurate analytical and imaging technique which can reconstruct the complex structure and composition of a specimen in three dimensions. Despite providing locally high spatial resolution, atom probe tomography suffers from global distortions due to a complex projection function between the specimen and detector which is different for each experiment and can change during a single run. To aid characterization of this projection function, this work demonstrates a method for the reverse projection of ions from an arbitrary projection surface in 3D space back to an atom probe tomography specimen surface. Experimental data from transmission electron microscopy tilt tomography are combined with point cloud surface reconstruction algorithms and finite element modelling to generate a mapping back to the original tip surface in a physically and experimentally motivated manner. As a case study, aluminium tips are imaged using transmission electron microscopy before and after atom probe tomography, and the specimen profiles used as input in surface reconstruction methods. This reconstruction method is a general procedure that can be used to generate mappings between a selected surface and a known tip shape using numerical solutions to the electrostatic equation, with quantitative solutions to the projection problem readily achievable in tens of minutes on a contemporary workstation.     

Curtailed light sheet microscopy for rapid imaging of macroscopic biological specimens

We study the feasibility of volume imaging from a few angular views/scans in a light sheet fluorescence microscopy. Two‐dimensional (2D) images (angular views) were acquired at an angular separation of 10° and volume images were constructed with merely 18, 9, and 6 views. We study the structural changes in a 5‐day old Zebrafish embryo labeled with Phalloidin TRITC that binds to F‐Actin of embryo cell. To collect the data, the specimen is rotated (for varying sampling angles Δθ) with respect to a fixed vertical axis passing through the volume‐of‐interest (yolk sac). In the proposed realization of selective plane illumination microscopy (SPIM) technique, the translation is completely avoided. Analysis shows rich structural information with marginal reduction in contrast. Comparison with the state‐of‐the‐art SPIM shows appreciable volume reconstruction (from an order less 2D scans) that may be good enough for rapid monitoring of macroscopic specimens. Microsc. Res. Tech. 79:455–458, 2016. © 2016 Wiley Periodicals, Inc.     

基于ART的光学过程层析成像扇束图像重建

光学过程层析成像是近几年发展起来的一种新型的光学测量技术，它源于医学CT的基本理论和方法，可认为是医学CT技术在工业过程监测领域的自然延伸和发展。然而，由于过程层析成像的被测对象为快速变化的工业过程，因此，其投影数据的数量比医学CT少得多，而实时性则要求更高。利用一种扇束扫描制式的光学传感器有利于提高光学过程层析成像的空间分辨率及测量精度，但在这种扫描制式下，引用医学CT的常见图像重建算法(如数据重排方法、反投影法和滤波反投影法等)却不适用或难以胜任工业检测的要求。为此提出了一种代数重建技术来提高光学过程层析成像的测量精度和速度。该算法不仅适用于少数投影数据的情况，也能使求解过程遍历几乎所有的图像像元，因此成像效果好和实时性较高，具有工程应用价值。     

Automatic seamless mosaicing of microscopic images: enhancing appearance with colour degradation compensation and wavelet-based blending

In order to observe the fine details of biomedical specimens, various kinds of high-magnification microscopes are used. However, they suffer from a limited field of view when visualizing highly magnified specimens. Image mosaicing techniques are necessary to integrate two or more partially overlapping images into one and make the whole specimen visible. In this study, we propose a new system that automatically creates panoramic images by mosaicing all the microscopic images acquired from a specimen. Not only does it effectively compensate for the congenital narrowness in microscopic views, but it also results in the mosaiced image containing as little distortion with respect to the originals as possible. The system consists of four main steps: (1) feature point extraction using multiscale wavelet analysis, (2) image matching based on feature points or by projection profile alignment, (3) colour difference adjustment and optical degradation compensation with a Gaussian-like model and (4) wavelet-based image blending. In addition to providing a precise alignment, the proposed system also takes into account the colour deviations and degradation in image mosaicing. The visible seam lines are eliminated after image blending. The experimental results show that the system performs well on differently stained image sequences and is effective on acquired images with large colour variations and degradation. It is expected to be a practical tool for microscopic image mosaicing.     

内—外置电极ECT传感器两相流成像仿真研究

本文利用仿真方法研究一种新型具有内置电极的矩形电容层析成像传感器。其由4个内置电极和12个外置电极组成。通过有限差分方法求得普适的矩形敏感图分布，并进一步对其进行处理得到针对这种特殊传感器的专门敏感图。对三种典型的物质分布进行了成像研究，并与无内置电极的12电极测量装置进行比较。计算结果显示了具有内置电极的传感器比无内置电极传感器具有更高的成像质量。     

基于差分吸收光谱技术的大气痕量气体重建算法

针对在一些不能获取大量投影数据或者投影分布不均匀的实际情况下,重建大气痕量气体的二维空间分布问题,采用代数迭代重建算法(ART)和同步迭代重建算法(SIRT)2种重建算法对其进行重建。通过数值模拟的方式对2种重建算法的重建效果进行了比较研究,并建立了多轴差分吸收光谱层析系统,进行了外场试验。通过计算机模拟和外场实验研究,从成像效果中可以看出,2种迭代算法均能在数据不完善的情况下,精确地重建出大气痕量气体的二维空间分布;在不同的模型及评价指标下,通过对2种重建算法的重建误差的比较,可以发现SIRT算法的重建误差要小于ART算法的重建误差,尤其在测量数据波动比较大、受干扰的情况下。     

Invariant classification of molecular views in electron micrographs

Biological macromolecules can exhibit many different orientations in electron microscopical preparations. In particular in vitreous-ice-embedded specimens, the number of different views can be high. Existing techniques of analysis require the alignment of the molecular views relative to one or more reference images with cross-correlation ("matched filtering") techniques and are somewhat unsatisfactory because of the high noise level and the large number of different views in such images. We here propose a method in which first rotation-, translation- and mirror-invariant functions are derived from the large set of input images. These functions are subsequently classified automatically using multivariate statistical classification techniques. The different molecular views in the images can therewith be found without bias, provided that a statistically significant number of copies of the views are present in the data set. The basic ideas are exemplified with realistic model data.     

Attenuation correction in confocal laser microscopes: a novel two-view approach

Confocal microscopy is a three‐dimensional (3D) imaging modality, but the specimen thickness that can be imaged is limited by depth‐dependent signal attenuation. Both software and hardware methods have been used to correct the attenuation in reconstructed images, but previous methods do not increase the image signal‐to‐noise ratio (SNR) using conventional specimen preparation and imaging. We present a practical two‐view method that increases the overall imaging depth, corrects signal attenuation and improves the SNR. This is achieved by a combination of slightly modified but conventional specimen preparation, image registration, montage synthesis and signal reconstruction methods. The specimen is mounted in a symmetrical manner between a pair of cover slips, rather than between a slide and a cover slip. It is imaged sequentially from both sides to generate two 3D image stacks from perspectives separated by approximately 180° with respect to the optical axis. An automated image registration algorithm performs a precise 3D alignment, and a model‐based minimum mean squared algorithm synthesizes a montage, combining the content of both the 3D views. Experiments with images of individual neurones contrasted with a space‐filling fluorescent dye in thick brain tissue slices produced precise 3D montages that are corrected for depth‐dependent signal attenuation. The SNR of the reconstructed image is maximized by the method, and it is significantly higher than in the single views after applying our attenuation model. We also compare our method with simpler two‐view reconstruction methods and quantify the SNR improvement. The reconstructed images are a more faithful qualitative visualization of the specimen's structure and are quantitatively more accurate, providing a more rigorous basis for automated image analysis.     

基于椭球包围盒的锥束CT三维加权重建算法

提高锥束CT大锥角圆轨道扫描下的重建图像质量一直是CT成像技术的重要研究方向。分析了圆轨道扫描下Radon空间的Z向数据缺失特点和FDK算法的灰度下降规律,提出了一种基于椭球包围盒的锥束CT三维加权重建算法。该算法无需重排,只需利用投影数据获取重建物体的最小包围盒,并根据其内接椭球的大小和空间位置自动生成三维加权函数,在反投影阶段加入该加权函数即可实现。该算法不会引入其他伪影,计算量增加很小,并且在中心层与FDK算法等价。仿真扫描实验表明,该算法显著提高了FDK算法的准确性,减小了锥角伪影。     

Intensity correction of fluorescent confocal laser scanning microscope images by mean-weight filtering

This paper addresses the problem of intensity correction of fluorescent confocal laser scanning microscope images. Confocal laser scanning microscope images are frequently used in medicine for obtaining 3D information about specimen structures by imaging a set of 2D cross sections and performing 3D volume reconstruction afterwards. However, 2D images acquired from fluorescent confocal laser scanning microscope images demonstrate significant intensity heterogeneity, for example, due to photo‐bleaching and fluorescent attenuation in depth. We developed an intensity heterogeneity correction technique that (a) adjusts the intensity heterogeneity of 2D images, (b) preserves fine structural details and (c) enhances image contrast, by performing spatially adaptive mean‐weight filtering. Our solution is obtained by formulating an optimization problem, followed by filter design and automated selection of filtering parameters. The proposed filtering method is experimentally compared with several existing techniques by using four quality metrics: contrast, intensity heterogeneity (entropy) in a low frequency domain, intensity distortion in a high frequency domain and saturation. Based on our experiments and the four quality metrics, the developed mean‐weight filtering outperforms other intensity correction methods by at least a factor of 1.5 when applied to fluorescent confocal laser scanning microscope images.     

Microtomography from limited projections in conventional TEM for 3D reconstruction of an intact cell

It is possible to generate three-dimensional reconstructions of whole, non-sectioned biological cells in conventional TEM using an 80 kV tungsten source. A TEM specimen stage was modified to accommodate a precise single-axis tilting mechanism controlled by a digital stepping motor interfaced to a computer. For image collection, a video camera was optically coupled to the TEM phosphorescent screen, and the video image was digitized by a frame buffer interfaced to a computer. Specimen tilt and projection image collection were fully computer-automated. This microtomography system design could be readily adapted for most TEMs. Image reconstruction was achieved through computation on projection images from limited tilts; typically less than thirty projection images were needed for a coarse 3D reconstruction. The iterative reconstruction algorithm used certain statistical assumptions about the distribution of image gray values. Since microtomography was performed on non-sectioned whole mount cells viewed under an 80 kV electron beam, methods of embedment-free specimen preparation with chemical fixation and extraction were employed. These methods were utilized successfully to permit good image formation of the entire cell mitotic nucleus a few micrometers in thickness. The 3D reconstruction of a single kidney cell mitotic nucleus was carried out and shown to produce a reasonable microtomogram of gross features like the condensed chromosomes.     

短裂纹模拟及其CT仿真投影获取方法研究

短裂纹尺寸一般都在几个微米到几十个微米之间,已低于目前工业CT的最小分辨率,无法被检测出来。基于CT仿真系统,利用结构实体几何模型,设计了一种短裂纹的模拟方法以及其仿真投影数据的获取方法。首先仿真高于实际尺寸数倍的短裂纹群,以达到CT仿真系统的扫描精度,扫描获取该短裂纹群的CT投影数据;之后把该图像数据利用像素合并法缩小到实际尺寸,然后把缩小的CT图像合并,此时便得到了实际尺寸的短裂纹群CT仿真投影图像。利用该结果可以进行下一步的CT重建和疲劳寿命分析等方面的研究。试验证明该方法能得到较为真实的短裂纹群CT投影数据。     

Three‐dimensional imaging of whole mouse models: comparing nondestructive X‐ray phase‐contrast micro‐CT with cryotome‐based planar epi‐illumination imaging

In this study, we compare two evolving techniques for obtaining high‐resolution 3D anatomical data of a mouse specimen. On the one hand, we investigate cryotome‐based planar epi‐illumination imaging (cryo‐imaging). On the other hand, we examine X‐ray phase‐contrast micro‐computed tomography (micro‐CT) using synchrotron radiation. Cryo‐imaging is a technique in which an electron multiplying charge coupled camera takes images of a cryo‐frozen specimen during the sectioning process. Subsequent image alignment and virtual stacking result in volumetric data. X‐ray phase‐contrast imaging is based on the minute refraction of X‐rays inside the specimen and features higher soft‐tissue contrast than conventional, attenuation‐based micro‐CT. To explore the potential of both techniques for studying whole mouse disease models, one mouse specimen was imaged using both techniques. Obtained data are compared visually and quantitatively, specifically with regard to the visibility of fine anatomical details. Internal structure of the mouse specimen is visible in great detail with both techniques and the study shows in particular that soft‐tissue contrast is strongly enhanced in the X‐ray phase images compared to the attenuation‐based images. This identifies phase‐contrast micro‐CT as a powerful tool for the study of small animal disease models.     

完全最小二乘下的康普顿散射图像重建

由康普顿散射能谱重建密度图像的问题是一个非线性的逆问题 ,这给重建带来很大困难。解决问题的一种方法是结合透射测量所得的衰减系数用以消除问题的非线性。但是 ,用这种方法所得到的投影矩阵却存在误差。为了减小这种误差对重建结果的影响 ,提出用完全最小二乘法来重建康普顿散射图像。数值模拟实验证明 ,这种方法极大地改善了重建图像的质量。     

Tomography experiment of an integrated circuit specimen using 3 MeV electrons in the transmission electron microscope

The possibility of utilizing high-energy electron tomography to characterize the micron-scale three dimensional (3D) structures of integrated circuits has been demonstrated experimentally. First, electron transmission through a tilted SiO(2) film was measured with an ultrahigh-voltage electron microscope (ultra-HVEM) and analyzed from the point of view of elastic scattering of electrons, showing that linear attenuation of the logarithmic electron transmission still holds valid for effective specimen thicknesses up to 5 microm under 2 MV accelerating voltages. Electron tomography of a micron-order thick integrated circuit specimen including the Cu/via interconnect was then tried with 3 MeV electrons in the ultra-HVEM. Serial projection images of the specimen tilted at different angles over the range of +/-90 degrees were acquired, and 3D reconstruction was performed with the images by means of the IMOD software package. Consequently, the 3D structures of the Cu lines, via and void, were revealed by cross sections and surface rendering.     

三维锥束FDK的一种新插值算法

三维锥束重建算法中,近似算法由于数学形式简单,实现容易,而且锥角较小时重建效果较好,所以,实际中广泛应用。在各种基于滤波反投影的近似算法中,FDK算法一直是实际应用中的主流。文章基于体素和晶胞的相似性,把晶胞的特点应用到体素中,提出FDK算法的一种改进方法,并用三维Shepp-Logan模型进行重建。实验结果表明,新方法重建的图像比传统FDK方法清晰,而且还有抑制噪声的作用。