On the freezing and identification of lipid monolayer 2-D arrays for cryoelectron microscopy

Lipid monolayers provide a convenient vehicle for the crystallization of biological macromolecules for 3-D electron microscopy. Although numerous examples of 3-D images from 2-D protein arrays have been described from negatively stained specimens, only six structures have been done from frozen-hydrated specimens. We describe here a method that makes high quality frozen-hydrated specimens of lipid monolayer arrays for cryoelectron microscopy. The method uses holey carbon films with patterned holes for monolayer recovery, blotting and plunge freezing to produce thin aqueous films which cover >90% of the available grid area. With this method, even specimens with relatively infrequent crystals can be screened using automated data collection techniques. Though developed for microscopic examination of 2-D arrays, the method may have wider application to the preparation of single particle specimens for 3-D image reconstruction.     

Largest contour segmentation: a tool for the localization of spots in confocal images

An accurate determination of the 3-D positions of multiple spots in images obtained by confocal microscopy is essential for the investigation of the spatial distribution of specific components or processes in biological specimens. The position of the centroid, as an estimator for the position of a spot, can be calculated on the basis of all voxels that belong to the domain of the spot. For this calculation a domain that defines which voxels belong to the spot must be delimited. To create a boundary for a domain we developed a 3-D image segmentation procedure: the largest contour segmentation (LCS). This procedure is based on an iterative region-growing procedure around each local maximum of intensity. By means of this procedure the position of each spot was determined accurately and automatically. Qualities of the procedure were evaluated by means of simulated test-images as well as 3-D images of real biological specimens.     

Three-dimensional imaging of monogenoidean sclerites by laser scanning confocal fluorescence microscopy

A nondestructive protocol for preparing specimens of Monogenoidea for both alpha-taxonomic studies and reconstruction of 3-dimensional structure is presented. Gomori's trichrome, a stain commonly used to prepare whole-mount specimens of monogenoids for taxonomic purposes, is used to provide fluorescence of genital spines, the copulatory organ, accessory piece, squamodisc, anchors, hooks, bars, and clamps under laser scanning confocal microscopy.     

Assessment of precision and accuracy of digital surface photogrammetry with the DSP 400 system]

The objective of the present study was to evaluate the precision and accuracy of facial anthropometric measurements obtained through digital 3-D surface photogrammetry with the DSP 400 system in comparison to traditional 2-D photogrammetry. Fifty plaster casts of cleft infants were imaged and 21 standard anthropometric measurements were obtained. For precision assessment the measurements were performed twice in a subsample. Accuracy was determined by comparison of direct measurements and indirect 2-D and 3-D image measurements. Precision of digital surface photogrammetry was almost as good as direct anthropometry and clearly better than 2-D photogrammetry. Measurements derived from 3-D images showed better congruence to direct measurements than from 2-D photos. Digital surface photogrammetry with the DSP 400 system is sufficiently precise and accurate for craniofacial anthropometric examinations.     

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.     

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.     

3-D Visualization and Quantitation of Microvessels in Transparent Human Colorectal Carcinoma

Microscopic analysis of tumor vasculature plays an important role in understanding the progression and malignancy of colorectal carcinoma. However, due to the geometry of blood vessels and their connections, standard microtome-based histology is limited in providing the spatial information of the vascular network with a 3-dimensional (3-D) continuum. To facilitate 3-D tissue analysis, we prepared transparent human colorectal biopsies by optical clearing for in-depth confocal microscopy with CD34 immunohistochemistry. Full-depth colons were obtained from colectomies performed for colorectal carcinoma. Specimens were prepared away from (control) and at the tumor site. Taking advantage of the transparent specimens, we acquired anatomic information up to 200 μm in depth for qualitative and quantitative analyses of the vasculature. Examples are given to illustrate: (1) the association between the tumor microstructure and vasculature in space, including the perivascular cuffs of tumor outgrowth, and (2) the difference between the 2-D and 3-D quantitation of microvessels. We also demonstrate that the optically cleared mucosa can be retrieved after 3-D microscopy to perform the standard microtome-based histology (H&E staining and immunohistochemistry) for systematic integration of the two tissue imaging methods. Overall, we established a new tumor histological approach to integrate 3-D imaging, illustration, and quantitation of human colonic microvessels in normal and cancerous specimens. This approach has significant promise to work with the standard histology to better characterize the tumor microenvironment in colorectal carcinoma.     

How Accurate Are the Fusion of Cone-Beam CT and 3-D Stereophotographic Images?

Background

Cone-beam Computed Tomography (CBCT) and stereophotography are two of the latest imaging modalities available for three-dimensional (3-D) visualization of craniofacial structures. However, CBCT provides only limited information on surface texture. This can be overcome by combining the bone images derived from CBCT with 3-D photographs. The objectives of this study were 1) to evaluate the feasibility of integrating 3-D Photos and CBCT images 2) to assess degree of error that may occur during the above processes and 3) to identify facial regions that would be most appropriate for 3-D image registration.

Methodology

CBCT scans and stereophotographic images from 29 patients were used for this study. Two 3-D images corresponding to the skin and bone were extracted from the CBCT data. The 3-D photo was superimposed on the CBCT skin image using relatively immobile areas of the face as a reference. 3-D colour maps were used to assess the accuracy of superimposition were distance differences between the CBCT and 3-D photo were recorded as the signed average and the Root Mean Square (RMS) error.

Principal Findings:

The signed average and RMS of the distance differences between the registered surfaces were −0.018 (±0.129) mm and 0.739 (±0.239) mm respectively. The most errors were found in areas surrounding the lips and the eyes, while minimal errors were noted in the forehead, root of the nose and zygoma.

Conclusions

CBCT and 3-D photographic data can be successfully fused with minimal errors. When compared to RMS, the signed average was found to under-represent the registration error. The virtual 3-D composite craniofacial models permit concurrent assessment of bone and soft tissues during diagnosis and treatment planning.     

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.     

Soft x-ray microscopy

Soft x-ray microscopes are beginning to provide information to complement that obtained from optical and electron microscopy. Soft x-ray microscopy can deliver 30-nm resolution images of hydrated cells up to approximately 10 microns thick, and efforts towards obtaining higher resolution are under way. Although living specimens cannot be studied readily except in single exposures, fixed samples can be imaged at high resolution, and flash-frozen specimens can be studied without chemical modification and without significant radiation damage. Tomography is being developed for 3-D imaging, and spectromicroscopy offers unique capabilities for biochemical mapping of unlabelled structures beyond those of gold and fluorescent labels. Currently, most soft x-ray microscopes operate at synchrotron radiation facilities, but laboratory-scale microscopes are being developed too.     

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.     

Real space refinement of acto-myosin structures from sectioned muscle

We have adapted a real space refinement protocol originally developed for high-resolution crystallographic analysis for use in fitting atomic models of actin filaments and myosin subfragment 1 (S1) to 3-D images of thin-sectioned, plastic-embedded whole muscle. The rationale for this effort is to obtain a refinement protocol that will optimize the fit of the model to the density obtained by electron microscopy and correct for poor geometry introduced during the manual fitting of a high-resolution atomic model into a lower resolution 3-D image. The starting atomic model consisted of a rigor acto-S1 model obtained by X-ray crystallography and helical reconstruction of electron micrographs. This model was rebuilt to fit 3-D images of rigor insect flight muscle at a resolution of 7 nm obtained by electron tomography and image averaging. Our highly constrained real space refinement resulted in modest improvements in the agreement of model and reconstruction but reduced the number of conflicting atomic contacts by 70% without loss of fit to the 3-D density. The methodology seems to be well suited to the derivation of stereochemically reasonable atomic models that are consistent with experimentally determined 3-D reconstructions computed from electron micrographs.     

InvertNet: a new paradigm for digital access to invertebrate collections

InvertNet, one of the three Thematic Collection Networks (TCNs) funded in the first round of the U.S. National Science Foundation's Advancing Digitization of Biological Collections (ADBC) program, is tasked with providing digital access to ~60 million specimens housed in 22 arthropod (primarily insect) collections at institutions distributed throughout the upper midwestern USA. The traditional workflow for insect collection digitization involves manually keying information from specimen labels into a database and attaching a unique identifier label to each specimen. This remains the dominant paradigm, despite some recent attempts to automate various steps in the process using more advanced technologies. InvertNet aims to develop improved semi-automated, high-throughput workflows for digitizing and providing access to invertebrate collections that balance the need for speed and cost-effectiveness with long-term preservation of specimens and accuracy of data capture. The proposed workflows build on recent methods for digitizing and providing access to high-quality images of multiple specimens (e.g., entire drawers of pinned insects) simultaneously. Limitations of previous approaches are discussed and possible solutions are proposed that incorporate advanced imaging and 3-D reconstruction technologies. InvertNet couples efficient digitization workflows with a highly robust network infrastructure capable of managing massive amounts of image data and related metadata and delivering high-quality images, including interactive 3-D reconstructions in real time via the Internet.     

基于VTK的医学图像三维可视化系统

医学图像的三维可视化可以通过可视化工具包（VTK）提供的API实现。VTK是医学图像可视化的开法工具包,它把可视化的算法封装起来,利用简单的代码生成所需图形。基于VTK的医学图像三维可视化系统阐述了如何借助VTKAPI读入二维医学图像序列、操作二维图像、重建三维图像以及进行三维图像可视化的全套方案,为临床医生的诊断、治疗提供了有益的途径。     

The Use of Electron Tomography for Structural Analysis of Disordered Protein Arrays

A method based on Fourier transforms is described for obtaining a 3-D reconstruction from a paracrystalline object with static disorder. The method is derived from the standard methods used in 3-D reconstruction of 2-D crystals except that all of the Fourier coefficients are used and not just the sampled data from the periodic lattice. Thus, not only is the spatially ordered part of the structure visualized in 3-D, but also the spatially disordered part. Application of the method to 3-D reconstructions of insect flight muscle is described as well as prospects for extension of the method to radiation-sensitive specimens.     

High-resolution three-dimensional-pQCT images can be an adequate basis for in-vivo microFE analysis of bone

Micro-finite element (microFE) models based on high-resolution images have enabled the calculation of elastic properties of trabecular bone in vitro. Recently, techniques have been developed to image trabecular bone structure in vivo, albeit at a lesser resolution. The present work studies the usefulness of such in-vivo images for microFE analyses, by comparing their microFE results to those of models based on high-resolution micro-CT (microCT) images. Fifteen specimens obtained from human femoral heads were imaged first with a 3D-pQCT scanner at 165 microns resolution and a second time with a microCT scanner at 56 microns resolution. A third set of images with a resolution of 165 microns was created by downscaling the microCT measurements. The microFE models were created directly from these images. Orthotropic elastic properties and the average tissue von Mises stress of the specimens were calculated from six FE-analyses per specimen. The results of the 165 microns models were compared to those of the 56 microns model, which was taken as the reference model. The results calculated from the pQCT-based models, correlated excellent with those calculated from the reference model for both moduli (R2 > 0.95) and for the average tissue von Mises stress (R2 > 0.83). Results calculated from the downscaled micro-CT models correlated even better with those of the reference models (R2 > 0.99 for the moduli and R2 > 0.96 for the average von Mises stress). In the case of the 3D-pQCT based models, however, the slopes of the regression lines were less than one and had to be corrected. The prediction of the Poisson's ratios was less accurate (R2 > 0.45 and R2 > 0.67) for the models based on 3D-pQCT and downscaled microCT images respectively). The fact that the results from the downscaled and original microCT images were nearly identical indicates that the need for a correction in the case of the 3D-pQCT measurements was not due to the voxel size of the images but due to a higher noise level and a lower contrast in these images, in combination with the application of a filtering procedure at 165 micron images. In summary: the results of microFE models based on in-vivo images of the 3D-pQCT can closely resemble those obtained from microFE models based on higher resolution microCT system.     

Combined method of whole mount and block-face imaging: Acquisition of 3D data of gene expression pattern from conventional in situ hybridization

Visualization of spatiotemporal expression of a gene of interest is a fundamental technique for analyzing the involvements of genes in organ development. In situ hybridization (ISH) is one of the most popular methods for visualizing gene expression. When conventional ISH is performed on sections or whole-mount specimens, the gene expression pattern is represented in 2-dimensional (2D) microscopic images or in the surface view of the specimen. To obtain 3-dimensional (3D) data of gene expression from conventional ISH, the “serial section method” has traditionally been employed. However, this method requires an extensive amount of time and labor because it requires researchers to collect a tremendous number of sections, label all sections by ISH, and image them before 3D reconstruction. Here, we proposed a rapid and low-cost 3D imaging method that can create 3D gene expression patterns from conventional ISH-labeled specimens. Our method consists of a combination of whole-mount ISH and Correlative Microscopy and Blockface imaging (CoMBI). The whole-mount ISH-labeled specimens were sliced using a microtome or cryostat, and all block-faces were imaged and used to reconstruct 3D images by CoMBI. The 3D data acquired using our method showed sufficient quality to analyze the morphology and gene expression patterns in the developing mouse heart. In addition, 2D microscopic images of the sections can be obtained when needed. Correlating 2D microscopic images and 3D data can help annotate gene expression patterns and understand the anatomy of developing organs. These results indicated that our method can be useful in the field of developmental biology.     

High-resolution cryo-electron microscopy on macromolecular complexes and cell organelles

Cryo-electron microscopy techniques and computational 3-D reconstruction of macromolecular assemblies are tightly linked tools in modern structural biology. This symbiosis has produced vast amounts of detailed information on the structure and function of biological macromolecules. Typically, one of two fundamentally different strategies is used depending on the specimens and their environment. A: 3-D reconstruction based on repetitive and structurally identical unit cells that allow for averaging, and B: tomographic 3-D reconstructions where tilt-series between approximately ±60 and ±70° at small angular increments are collected from highly complex and flexible structures that are beyond averaging procedures, at least during the first round of 3-D reconstruction. Strategies of group A are averaging-based procedures and collect large number of 2-D projections at different angles that are computationally aligned, averaged together, and back-projected in 3-D space to reach a most complete 3-D dataset with high resolution, today often down to atomic detail. Evidently, success relies on structurally repetitive particles and an aligning procedure that unambiguously determines the angular relationship of all 2-D projections with respect to each other. The alignment procedure of small particles may rely on their packing into a regular array such as a 2-D crystal, an icosahedral (viral) particle, or a helical assembly. Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern. In contrast, tomographic 3-D reconstruction procedures (group B) do not rely on averaging, but collect an entire dataset from the very same structure of interest. Data acquisition requires collecting a large series of tilted projections at angular increments of 1–2° or less and a tilt range of ±60° or more. Accordingly, tomographic data collection exposes its specimens to a large electron dose, which is particularly problematic for frozen-hydrated samples. Currently, cryo-electron tomography is a rapidly emerging technology, on one end driven by the newest developments of hardware such as super-stabile microscopy stages as well as the latest generation of direct electron detectors and cameras. On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data. Here, we will review the status quo of cryo-electron microscopy and discuss the future of cellular cryo-electron tomography from data collection to data analysis, CTF-correction of tilt-series, post-tomographic sub-volume averaging, and 3-D particle classification. We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree.     

A micro-computed tomography study of the trabecular bone structure in the femoral head

The goal of this study was to characterize the trabecular microarchitecture of the femoral head using micro-computed tomography (ICT). Femoral head specimens were obtained from subjects following total hip replacement. Cylindrical cores from the specimens were scanned to obtain 3-D images with an isotropic resolution of 26 Im. Bone structural parameters were evaluated on a per millimeter basis: relative bone volume (BV/TV), trabecular number (Tb.N), thickness (Tb.Th) and separation (Tb.Sp), structure model index (SMI), and connectivity (Conn.D). The ICT data show that the first two millimeters, starting at the joint surface, are characterized by more plate-like trabeculae, and are significantly denser than the underlying trabecular bone. Regional differences in the trabecular architecture reveal that the superior pole has significantly higher BV/TV, Tb.N and Tb.Th values, with lower Tb.Sp compared to the inferior and side poles. Because subchondral bone is essential in the load attenuation of joints, the difference in bone structure between the subchondral and trabecular bone might arise from the different functions each have within joint-forming bones. The denser trabecular structure of the superior pole as compared to the inferior pole can be interpreted as a functional adaptation to higher loading in this area.     

Feasibility of using orthogonal fluoroscopic images to measure in vivo joint kinematics

Accurately determining in vivo knee kinematics is still a challenge in biomedical engineering. This paper presents an imaging technique using two orthogonal images to measure 6 degree-of-freedom (DOF) knee kinematics during weight-bearing flexion. Using this technique, orthogonal images of the knee were captured using a 3-D fluoroscope at different flexion angles during weight-bearing flexion. The two orthogonal images uniquely characterized the knee position at the specific flexion angle. A virtual fluoroscope was then created in solid modeling software and was used to reproduce the relative positions of the orthogonal images and X-ray sources of the 3-D fluoroscope during the actual imaging procedure. Two virtual cameras in the software were used to represent the X-ray sources. The 3-D computer model of the knee was then introduced into the virtual fluoroscope and was projected onto the orthogonal images by the two virtual cameras. By matching the projections of the knee model to the orthogonal images of the knee obtained during weight-bearing flexion, the knee kinematics in 6 DOF were determined. Using regularly shaped objects with known positions and orientations, this technique was shown to have an accuracy of 0.1 mm and 0.1 deg in determining the positions and orientations of the objects, respectively.