A COMPUTER GRAPHICS PROGRAM FOR THE THREE-DIMENSIONAL RECONSTRUCTION OF PLANT ORGANS FROM SERIAL SECTIONS

Morphological and anatomical studies have long utilized qualitative means to reconstruct 3-dimensional transparent images from serial sections. With the advent of computer graphics systems, more accurate images can be constructed to visualize growing plant parts in transparent 3-D. We have developed a computer program which allows such reconstruction from stained serial sections. Advantages include 3-dimensional imaging without the use of stereo pairs and the ability to rotate an image to see all angles.     

3-D image analysis system and megakaryocyte quantitation

A quasi-automatic computer image analysis system has been developed for 3-D reconstruction of stained serial sections and implemented on an IBAS system. Some new automatic image analysis techniques have been designed and incorporated into the system. For image segmentation, a transition region determination based thresholding method is introduced. Neither histogram calculation nor empirical parameters are needed in the automatic threshold selection. A two step 3-D reconstruction procedure--symbolic and pictorial reconstructions--is designed to improve the flexibility and the computational capability of the system. The global level registration and local level registration are separated. The former consists of establishing the relationship among a large numbers of profile pairs dispersed in adjacent sections. A pattern matching method based on pattern recognition principles is devised to exploit the information about the statistical character of mismatch caused by deformation of sections and about the relationship of nearby objects. For the latter, an equivalent elliptical approximation method based on the physical theory of the rotation of rigid bodies is proposed. The system has been used for 3-D reconstruction and quantitation of megakaryocytes in human bone marrow tissue. Features about individual 3-D megakaryocyte cell and the spatial distribution of megakaryocytes are determined. The latter is a new contribution to megakaryocyte quantitation and is not possible by using conventional stereologic techniques. These experimental results have demonstrated the ability of the system to perform quantitative analysis.     

A New Histochemical Double-Stain Method Using Three-Dimensional Analysis with Confocal laser Scanning Microscopy

We describe a new technique for immunohistochemical and enzyme-histochemi-cal double staining using confocal laser scanning microscopy in the reflection mode. As an example, we investigated the immunoreactivity for Spot 35-calbindin-D_28K, a vitamin D-dependent calcium binding protein, and the enzyme activity for ma⁺-ATPase in the rat kidney. The lead precipitation method for Ca²⁺-ATPase was initially used to process kidney slices. Each specimen was then dehydrated and embedded in a water soluble resin. Thin sections were cut from the resin block, and an indirect immunocolloidal gold method with silver enhancement for Spot 35-calbindin-D_28K antigen was carried out on the glass slides. Results were then observed by confocal laser scanning microscopy in the reflection mode. The three-dimensional distribution of the reaction products was detected by serial optic slice images. Lead phosphate particles, which represented the location of Ca²⁺-ATPase, were distributed deep in the section. The most intense signals from the silver partkles were detected from the surface slice of the section. A stereoscopic image generated from the serial optic slices clearly showed the differences in their distribution.     

Analysis by Confocal Microscopy of the Structure of Cambium in the Hardwood Kalopanax pictus

An understanding of the morphology and the developmental changesin the shapes and dimensions of cambial cells requires three-dimensional(3-D) analysis of thick slices of tissue. We devised a simpleprotocol using confocal laser scanning microscopy (CLSM), withsafranin and acridine orange as fluorescent dyes and glycerolas the clearing and mounting medium, to examine the 3-D structureof the dormant cambium in Kalopanax pictus, a ring-porous hardwood.Optical sections and high contrast images provided clear informationabout the shapes and nuclear status of cambial cells, whichhave previously been difficult to determine using conventionalmicroscopy. The axially-oriented cambial cells were found tovary in shape, in particular around the rays, and were not alwaystypically fusiform. We evaluated the reliability of our methodby comparing results with those of a parallel study of the samematerial by standard analysis of serial sections of epoxy-embeddedspecimens. The images of optical sections obtained by CLSM wereof high quality and similar to images obtained by conventionallight microscopy of semi-thin mechanical sections. Use of theconfocal microscope provided a quick and easy method for visualizationof the structure of the cambium in thick hand-cut sections andfor studies of the developmental changes in cells from the cambiumto the xylem. Copyright 2000 Annals of Botany Company Confocal laser scanning microscopy, Kalopanax pictus, three-dimensional reconstruction, vascular cambium     

生物组织显微结构三维重建的灰度阴影立体图对显示技术

本文描述了一种新的显示三维重建模型的方法—灰度阴影立体图对方法(shading-stereo-pair method).该方法根据连续切片三维重建的灰度阴影法和立体图对法的显示原理,导出了生成立体图对的视差公式.在灰度阴影法三维重建过程中,根据各切片所处的深度和其本身的厚度,按视差公式计算出它们在图对上的位移量并进行水平方向的移动,经过对各切片的叠加和重组,最后生成一幅具有视差信息的灰度阴影立体图对.在体视仪下观察,即可看到重建模型的三维实体图象.最后还讨论了进一步提高图象质量和改善观察效果所必须采取的措施.     

生物显微结构连续切片图象的象素地址矢量表达和三维重建信息加工

本文主要介绍一种适用于生物显微结构连续切片三维重建的图象表达方法——象素地址矢量法及其相应的图象信息加工方法:三维重建,三维模型绕任选空间轴旋转和用软件重新剖切.与常用的图象矩阵表达法相比,可以较多地节省序列图象所占的数据容量,便于在微机系统上对多种形态的显微结构进行三维重建信息加工.当对较大的图象进行分解与整合处理时,还避免了采用图象矩阵表达法可能遇到的边界问题.由于数据容量的减少,提高了图象处理的速度.这种图象表达及信息加工方法对包括生物活体时间序列图象在内的多种序列图象加工可能都是适用的.     

Validation study of skull three-dimensional computerized tomography measurements

Recent advances in imaging have led to high-resolution computerized tomography (CT) scanning with exquisitely detailed slice images of the skull and three-dimensional (3-D) surface reconstructions using computer software. It is possible to use CT scans to acquire morphologic information about the skull in a convenient digital form and to derive 3-D measurements from surface reconstruction images. Unfortunately, no effort has been made to date to test the validity of these measurements on laboratory specimens, and no compelling evidence is available from phantom studies to indicate the nature and magnitude of the errors inherent in the measurement technique. We have performed a pilot study to quantify the morphology of the skull based on surface features that can be found in CT scans and 3-D reconstructions. Comparative measurements were obtained from five skulls (two normal and three with dysmorphology) with calipers and a 3-D electromagnetic digitizer. These measurements were statistically compared with those based on original CT scan slices and reformatted 3-D images. It is concluded that 3D-CT measurement techniques are superior to those in which measurements are obtained directly from the original CT slices; 3-D CT methods, however, must be significantly improved before measurements based on these techniques can be used in studies that require a high degree of precision. The results are used to indicate the most fruitful areas of future study.     

A microcomputer-based algorithm for digitized image simplification and information extraction

Spatial analysis of objects often requires significant image simplification prior to information extraction and application of a decision-making algorithm. Much decision making based on images (e.g., histologic diagnoses) requires identifying patterns in complex backgrounds (image simplification) and comparison of those patterns to other patterns (decision making). Automated extraction of information from images commonly requires the extraction system to recognize edges (contours) of structures and their internal discontinuities (such as gradations in density) and to selectively suppress irrelevant data in order to conserve memory and speed computation; data from homogeneous image areas occupy memory, but are noncontributory or redundant. This paper describes the development of a microcomputer-based algorithm that deletes all homogeneous information from overlaid digitized images, generating contours in the place of nonhomogeneities. Contours corresponding to different areas or objects depend on color differences between an object and its surroundings. Any set of contours can be deleted almost instantaneously, leaving only those of interest. Contours can be highlighted by an operator-driven interactive process if desired and can be deleted and retrieved until an appropriate image is obtained. This contour-generating and image-simplification algorithm facilitates three-dimensional reconstruction of an object from serial images by reducing the number of calculations required and yielding a cleaner final image.     

Three-dimensional evaluation of the spinal local neural network revealed by the high-voltage electron microscopy: a double immunohistochemical study

Three-dimensional (3-D) analysis of anatomical ultrastructures is important in biological research. However, 3-D image analysis on exact serial sets of ultra-thin sections from biological specimens is very difficult to achieve, and limited information can be obtained by 3-D reconstruction from these sections due to the small area that can be reconstructed. On the other hand, the high-penetration power of electrons by an ultra-high accelerating voltage enables thick sections of biological specimens to be examined. High-voltage electron microscopy (HVEM) is particularly useful for 3-D analysis of the central nervous system because considerably thick sections can be observed at the ultrastructure level. Here, we applied HVEM tomography assisted by light microscopy to a study of the 3-D chemical neuroanatomy of the rat lower spinal cord annotated by double-labeling immunohistochemistry. This powerful methodology is useful for studying molecular and/or chemical neuroanatomy at the 3-D ultrastructural level.     

Electron microscope analysis of tissue components identified and located by computer-assisted 3-D reconstructions: ultrastructural segmentation of the developing human proximal tubule

A method is described for ultrastructural analysis of renal tubules after precise identification of tubule segments by computerized 3-D reconstruction at the light microscope level. Semithin serial sections were cut of entire nephrons and 3-D coordinate information was obtained by digitization of tubule cross sections in the semithin sections. With the aid of the computer the tubule axis was traced from one section to the other. Precise lengths and positions of the tubules in three dimensions were calculated and stereoscopic images generated. The method was used to analyze the 3-D structure of developing human nephrons, and the ultrastructural development of the proximal tubule. Ultrastructural segmentation of the proximal tubule was demonstrated in the human fetal nephron in developmental stage IV.     

A Reference-Free Method for Brightness Compensation and Contrast Enhancement of Micrographs of Serial Sections

Three-dimensional (3D) reconstruction of an organ or tissue from a stack of histologic serial sections provides valuable morphological information. The procedure includes section preparation of the organ or tissue, micrographs acquisition, image registration, 3D reconstruction, and visualization. However, the brightness and contrast through the image stack may not be consistent due to imperfections in the staining procedure, which may cause difficulties in micro-structure identification using virtual sections, region segmentation, automatic target tracing, etc. In the present study, a reference-free method, Sequential Histogram Fitting Algorithm (SHFA), is therefore developed for adjusting the severe and irregular variance of brightness and contrast within the image stack. To apply the SHFA, the gray value histograms of individual images are first calculated over the entire image stack and a set of landmark gray values are chosen. Then the histograms are transformed so that there are no abrupt changes in progressing through the stack. Finally, the pixel gray values of the original images are transformed into the desired ones based on the relationship between the original and the transformed histograms. The SHFA is tested on an image stacks from mouse kidney sections stained with toluidine blue, and captured by a slide scanner. As results, the images through the entire stack reveal homogenous brightness and consistent contrast. In addition, subtle color differences in the tissue are well preserved so that the morphological details can be recognized, even in virtual sections. In conclusion, compared with the existing histogram-based methods, the present study provides a practical method suitable for compensating brightness, and improving contrast of images derived from a large number of serial sections of biological organ.     

利用共聚焦显微镜系统进行视觉显微结构的三维重建

本文介绍利用共聚焦激光扫描显微镜系统进行的三种动物视觉显微结构的三维重建.所重建的对象为鸽视顶盖的神经元,蜻蜒小眼的晶锥和蟾蜍两个视顶盖之间的纤维联接结构.通过对约60μm厚的样品的共聚焦激光扫描,得到了1和3μm厚的连续光学切面的图象.利用计算机对这些图象进行三维重建得到的模型富有实体感和体视感,特别是以荧光染料标记的样品其三维重建结果比预料的好.三维重建的结果首次展示了这三种视觉显微结构的三维形态,这对进一步研究视觉显微结构的定量形态学和结构功能关系有重要意义,特别是这种装置能研究活组织的三维构型.对该系统的原理和优良性能也作了介绍.     

A statistical approach to computer processing of cryo-electron microscope images: virion classification and 3-D reconstruction

The scattering density of the virus is represented as a truncated weighted sum of orthonormal basis functions in spherical coordinates, where the angular dependence of each basis function has icosahedral symmetry. A statistical model of the image formation process is proposed and the maximum likelihood estimation method computed by an expectation-maximization algorithm is used to estimate the weights in the sum and thereby compute a 3-D reconstruction of the virus particle. If multiple types of virus particle are represented in the boxed images then multiple 3-D reconstructions are computed simultaneously without first requiring that the type of particle shown in each boxed image be determined. Examples of the procedure are described for viruses with known structure: (1). 3-D reconstruction of Flockhouse Virus from experimental images, (2). 3-D reconstruction of the capsid of Nudaurelia Omega Capensis Virus from synthetic images, and (3). 3-D reconstruction of both the capsid and the procapsid of Nudaurelia Omega Capensis Virus from a mixture of unclassified synthetic images.     

Finite element simulation of skeletal muscular structures obtained from images of histological serial sections

In this study, we present a method for the three-dimensional reconstruction of objects obtained from histological serial sections (exemplified by those of a pennate striated skeletal muscle) and its application to the finite element method. A hyperelastic material model is used for modeling biological soft tissue. The reconstruction process relies on the direct construction of a volumetric mesh using an octree approach which leads to a stable finite element method. Stability can be expressed in the spectral matrix condition number. To visualize stress patterns within the underlying anatomy the simulation results are projected onto images of the histological scenario.     

Automated image-based cytometry with fluorescence-stained specimens

The combination of digitized microscopy, algorithms for object recognition and fluorescent labeling is a promising approach for reliable, quick, automated and cost-effective screening of clinical specimens. We describe two conceptually different algorithms for detecting objects in fluorescence microscopic images. One, which is partially automated, compares a mask that represents a typical object with every position in the image; the other, which is fully automated, calculates threshold intensities to segment the image into regions of objects and background. Applications of the algorithms in conjunction with a prototype image-based cytometer are demonstrated for determining the DNA ploidy distribution of cultured human endometrial cells and determining the DNA ploidy distribution and the fraction of cells expressing the E6 antigen of human papilloma virus serotypes 16 and 18 in a PAP smear. The encouraging results from this study suggest that automated image-based cytometry utilizing fluorescent stains will be a valuable asset for clinical screening.     

Three-dimensional computer reconstructions from serial sections of cell nuclei

The analysis of ultrathin serial sections as 3-dimensional (3D) information requires interpretation and display of a large amount of data. We suggest a simple way to solve this problem; it permits presentation of a series of sections as a 3D color image of good quality. It involves a picture system with specialized hardware and software written for this purpose. 3D images of cellular organelles have been drawn either by manually defining the contour of the objects or by thresholding of the volumes in the structures. These 2 methods allow rapid drawing of the image on the screen. It is possible to determine the position, shape and size of 3D structures. This interactive system allows the user to choose between several options: colors, removal of parts of the object, and cutout.     

Fast and Accurate Registration of the Proximal Femurs in Bilateral Hip Joint Images by Using the Random Sub-Sample Points

Background and ObjectiveAs is known, point clouds representing the objects are frequently used in object registration. Although the objects can be registered by using all the points in the corresponding point clouds of the objects, the registration process can also be achieved with a smaller number of the landmark points selected from the entire point clouds of the objects. This paper introduces a research study focusing on the fast and accurate rigid registration of the bilateral proximal femurs in bilateral hip joint images by using the random sub-sample points. For this purpose, Random Point Sub-sampling (RPS) was analyzed and the reduced point sets were used for an accurate registration of the bilateral proximal femurs in coronal hip joint magnetic resonance imaging (MRI) slices.MethodsIn registration, bilateral proximal femurs in MRI slices were registered rigidly by performing a process consisting of three main phases named as MR image preprocessing, proximal femur registration over the random sub-sample points and MR image postprocessing. In the stage of the MR image preprocessing, segmentation maps of the bilateral proximal femurs are obtained as region of interest (RoI) images from the entire MRI slices and then, the edge maps of the segmented proximal femurs are extracted. In the registration phase, the edge maps describing the proximal femur surfaces are represented as point clouds initially. Thereafter, the RPS is performed on the proximal femur point clouds and the number of points representing the proximal femurs is reduced at different ratios. For the registration of the point clouds, the Iterative Closest Point (ICP) algorithm is performed on the reduced sets of points. Finally, the registration procedures are completed by performing MR image postprocessing on the registered proximal femur images.ResultsIn performance evaluation tests performed on healthy and pathological proximal femurs in 13 bilateral coronal hip joint MRI slices of 13 Legg-Calve-Perthes disease (LCPD) patients, bilateral proximal femurs were successfully registered with very small error rates by using the reduced set of points obtained via the RPS and promising results were achieved. The minimum error rate was observed at RPS rate of 30% as the value of 0.41 (±0.31)% on all over the bilateral proximal femurs evaluated. When the range of RPS rate of 20-30% is considered as the reference, the elapsed time in registration can be reduced by almost 30-40% compared to the case where all the proximal femur points were included in registration. Additionally, it was observed that the RPS rate should be selected as at least 25% to achieve a successful registration with an error rate below 1%.ConclusionIt was concluded from the observed results that a more successful and faster registration can be accomplished by selecting fewer points randomly from the point sets of proximal femurs instead of using all the points describing the proximal femurs. Not only an accurate registration with low error rates was performed, but also a faster registration process was performed by means of the limited number of points that are sub-sampled randomly from the whole point sets.     

Invertebrate myosin filament: Parallel subfilament arrangement in the wall of solid filaments from the honeybee, Apis mellifica

Transverse serial sections (100-140 nm thick) of solid myosin filaments of the honeybee, Apis mellifica, were photographed in a JEM-200 electron microscope at 200 kV. The images were digitized and computer processed by rotational filtering. 87% of the myosin filaments showed 6-fold symmetry in their power spectra, confirming the results of earlier works (Beinbrech et al., 1988, 1991). To determine if the subfilaments were arranged parallel to the filament backbone, two methods were used. First, the three images of each myosin filament in the three serial sections were superimposed. 85% of the resulting images showed a strong peak for 6-fold symmetry and the averaged images showed 6 pairs of subfilaments, which gives evidence for parallel arrangement of the subfilaments relative to the filament axis. This result was confirmed by the second method in which a 3-dimensional reconstruction was made. An average image was made from the images of the same 17 myosin filaments from each of the three sections. The data for the 3-dimensional reconstruction were collected by tracing the outlines of the structures in the three successive sections. The resulting stereo image shows a parallel arrangement of the subfilaments.     

Volumic patient-specific reconstruction of muscular system based on a reduced dataset of medical images

Three-dimensional mechanical modelling of muscles is essential for various biomechanical applications and clinical evaluation, but it requires a tedious manual processing of numerous images. A muscle reconstruction method is presented based on a reduced set of images to generate an approximate parametric object from basic dimensions of muscle contours. A regular volumic mesh is constructed based on this parametric object. The approximate object and the corresponding mesh are deformed to fit the exact muscles contours yielding patient-specific geometry. Evaluation was performed by comparison of geometry to that obtained by contouring all computed tomography (CT) slices, and by quantification of the mesh quality criteria. Muscle fatty infiltration was estimated using a threshold between fat and muscle. Volumic fat index (VFI) of a muscle was computed using first all the complete CT scan slices containing the muscle (VFI(ref)) and a second time only the slices used for reconstruction (VFI(recons)). Mean volume error estimation was 2.6% and hexahedron meshes fulfilled quality criteria. VFI(recons) respect the individual variation of fat content.     

3-D reconstruction of early preimplantation mouse embryo

Method of 3-D reconstruction approaches for early mouse embryo in preimplantation stages was modified. The developed technique is based on application of light microscopy of serial thin sections and well known soft operating. The designed method enabled us 1) to get serial sections of a single mouse embryo; 2) to create an orthogonal system independent on the sample for orientation of virtual sections. The adequacy of 3-DR protocol was checked on reconstruction of air bubbles embedded in epoxy resin as a model of sphere.