High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (μCT).High resolution μCT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples^{1, 2}. As opposed to electron tomography, the μCT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, μCT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods^{3, 4}, which are both time consuming and destructive. Sample conditioning and positioning in μCT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180° or 360°between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms^5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.     

Evolving technologies for growing,imaging and analyzing 3D root system architecture of crop plants

A plant's ability to maintain or improve its yield under limiting conditions,such as nutrient de ficiency or drought,can be strongly in fluenced by root system architecture(RSA),the three-dimensional distribution of the different root types in the soil. The ability to image,track and quantify these root system attributes in a dynamic fashion is a useful tool in assessing desirable genetic and physiological root traits. Recent advances in imaging technology and phenotyping software have resulted in substantive progress in describing and quantifying RSA. We have designed a hydroponic growth system which retains the three-dimensional RSA of the plant root system,while allowing for aeration,solution replenishment and the imposition of nutrient treatments,as well as high-quality imaging of the root system. The simplicity and flexibility of the system allows for modi fications tailored to the RSA of different crop species and improved throughput. This paper details the recent improvements and innovations in our root growth and imaging system which allows for greater image sensitivity(detection of fine roots and other root details),higher ef ficiency,and a broad array of growing conditions for plants that more closely mimic those found under field conditions.     

Registration of 6-DOFs electrogoniometry and CT medical imaging for 3D joint modeling

The paper describes a method in which two data-collecting systems, medical imaging and electrogoniometry, are combined to allow the accurate and simultaneous modeling of both the spatial kinematics and the morphological surface of a particular joint. The joint of interest (JOI) is attached to a Plexiglas jig that includes four metallic markers defining a local reference system (R(GONIO)) for the kinematics data. Volumetric data of the JOI and the R(GONIO) markers are collected from medical imaging. The spatial location and orientation of the markers in the global reference system (R(CT)) of the medical-imaging environment are obtained by applying object-recognition and classification methods on the image dataset. Segmentation and 3D isosurfacing of the JOI are performed to produce a 3D model including two anatomical objects-the proximal and distal JOI segments. After imaging, one end of a custom-made 3D electrogoniometer is attached to the distal segment of the JOI, and the other end is placed at the R(GONIO) origin; the JOI is displaced and the spatial kinematics data is recorded by the goniometer. After recording, data registration from R(GONIO) to R(CT) occurred prior to simulation. Data analysis was performed using both joint coordinate system (JCS) and instantaneous helical axis (IHA).Finally, the 3D joint model is simulated in real time using the experimental kinematics data. The system is integrated into a computer graphics interface, allowing free manipulation of the 3D scene.The overall accuracy of the method has been validated with two other kinematics data collection methods including a 3D digitizer and interpolation of the kinematics data from discrete positions obtained from medical imaging. Validation has been performed on both superior and inferior radio-ulna joints (i.e. prono-supination motion). Maximal RMS error was 1 degrees and 1.2mm on the helical axis rotation and translation, respectively. Prono-supination of the forearm showed a total rotation of 132 degrees for 0.8mm of translation. The method reproducibility using JCS parameters was in average 1 degrees (maximal deviation=2 degrees ) for rotation, and 1mm (maximal deviation=2mm) for translation. In vitro experiments have been performed on both knee joint and ankle joint. Averaged JCS parameters for the knee were 109 degrees, 17 degrees and 4 degrees for flexion, internal rotation and abduction, respectively. Averaged maximal translation values for the knee were 12, 3 and 4mm posteriorly, medially and proximally, respectively. Averaged JCS parameters for the ankle were 43 degrees, 9 degrees and 3 degrees for plantarflexion, adduction and internal rotation, respectively. Averaged maximal translation values for the ankle were 4, 2 and 1mm anteriorly, medially and proximally, respectively.     

3D imaging of telomeres and nuclear architecture: An emerging tool of 3D nano‐morphology‐based diagnosis

Patient samples are evaluated by experienced pathologists whose diagnosis guides treating physicians. Pathological diagnoses are complex and often assisted by the application of specific tissue markers. However, cases still exist where pathologists cannot distinguish between closely related entities or determine the aggressiveness of the disease they identify under the microscope. This is due to the absence of reliable markers that define diagnostic subgroups in several cancers. Three‐dimensional (3D) imaging of nuclear telomere signatures is emerging as a new tool that may change this situation offering new opportunities to the patients. This article will review current and future avenues in the assessment of diagnostic patient samples. J. Cell. Physiol. 226: 859–867, 2011. © 2010 Wiley‐Liss, Inc.     

Fully Automatic Localization and Segmentation of 3D Vertebral Bodies from CT/MR Images via a Learning-Based Method

In this paper, we address the problems of fully automatic localization and segmentation of 3D vertebral bodies from CT/MR images. We propose a learning-based, unified random forest regression and classification framework to tackle these two problems. More specifically, in the first stage, the localization of 3D vertebral bodies is solved with random forest regression where we aggregate the votes from a set of randomly sampled image patches to get a probability map of the center of a target vertebral body in a given image. The resultant probability map is then further regularized by Hidden Markov Model (HMM) to eliminate potential ambiguity caused by the neighboring vertebral bodies. The output from the first stage allows us to define a region of interest (ROI) for the segmentation step, where we use random forest classification to estimate the likelihood of a voxel in the ROI being foreground or background. The estimated likelihood is combined with the prior probability, which is learned from a set of training data, to get the posterior probability of the voxel. The segmentation of the target vertebral body is then done by a binary thresholding of the estimated probability. We evaluated the present approach on two openly available datasets: 1) 3D T2-weighted spine MR images from 23 patients and 2) 3D spine CT images from 10 patients. Taking manual segmentation as the ground truth (each MR image contains at least 7 vertebral bodies from T11 to L5 and each CT image contains 5 vertebral bodies from L1 to L5), we evaluated the present approach with leave-one-out experiments. Specifically, for the T2-weighted MR images, we achieved for localization a mean error of 1.6 mm, and for segmentation a mean Dice metric of 88.7% and a mean surface distance of 1.5 mm, respectively. For the CT images we achieved for localization a mean error of 1.9 mm, and for segmentation a mean Dice metric of 91.0% and a mean surface distance of 0.9 mm, respectively.     

植物三维染色质构型研究进展

染色质在细胞核内的缠绕、折叠及其在细胞核内的空间排布是真核生物染色质构型的主要特征。在经典DNA探针荧光原位杂交显微观察的基础上,基于新一代测序技术的Hi-C及ChIA-PET染色质构型捕获技术已经被广泛应用于动物及植物细胞核染色质构型的研究中,并以新的角度定义了包括:染色体(质)域(chromosome territory)、A/B染色质区室(compartment A/B)、拓扑偶联结构域(topological associated domains,TADs)、染色质环(chromatin loops)等在内的多个更为精细的染色质构型。利用以上两种主流技术,越来越多的植物物种染色质构型特征被鉴定、分析和比较。本文系统分析和总结了近年来以植物细胞为模型的细胞核染色构型领域取得的重要成果,包括各级染色质构型特征的组成、建立机制和主要影响因素等。在此基础上,分析了目前研究植物染色质构型技术的瓶颈和突破性的技术进展,并对后续研究主要关注的问题和研究内容进行了展望,以期为相关领域的研究提供更多的理论参考和依据。     

Breast volume assessment based on 3D surface geometry: verification of the method using MR imaging

Differences in breast volume and contour are subjectively estimated by surgeons. 3D surface imaging using 3D scanners provides objective breast volume quantification, but precision and accuracy of the method requires verification. Breast volumes of five test individuals were assessed using a 3D surface scanner. Magnetic resonance imaging (MRI) reference volumes were obtained to verify and compare the 3D scan measurements. The anatomical thorax wall curvature was segmented using MRI data and compared to the interpolated curvature of the posterior breast volume delimitation of 3D scan data. MRI showed higher measurement precision, mean deviation (expressed as percentage of volume) of 1.10+/-0.34% compared to 1.63+/-0.53% for the 3D scanner. Mean MRI [right (left) breasts: 638 (629)+/-143 (138) cc] and 3D scan [right (left) breasts: 493 (497)+/-112 (116) cc] breast volumes significantly correlated [right (left) breasts: r=0.982 (0.977), p=0.003 (0.004)]. The posterior thorax wall of the 3D scan model showed high agreement with the MRI thorax wall curvature [mean positive (negative) deviation: 0.33 (-0.17)+/-0.37 cm]. High correspondence and correlation of 3D scan data with MRI-based verifications support 3D surface imaging as sufficiently precise and accurate for breast volume measurements.     

RNA gradients: Shapers of 3D genome architecture

A growing body of evidence points to a role of nuclear RNAs (nucRNAs) in shaping the three-dimensional (3D) architecture of the genome within the nucleus of a eukaryotic cell. nucRNAs are non-homogeneously distributed within the nucleus where they can form global and local gradients that might contribute to instructing the formation and coordinating the function of different types of 3D genome structures. In this article, we highlight the available literature supporting a role of nucRNAs as 3D genome shapers and propose that nucRNA gradients are key mediators of genome structure and function.     

3D immuno-confocal image reconstruction of fibroblast cytoskeleton and nucleus architecture

Computational models of cellular structures generally rely on simplifying approximations and assumptions that limit biological accuracy. This study presents a comprehensive image processing pipeline for creating unified three-dimensional (3D) reconstructions of the cell cytoskeletal networks and nuclei. Confocal image stacks of these cellular structures were reconstructed to 3D isosurfaces (Imaris), then tessellations were simplified to reduce the number of elements in initial meshes by applying quadric edge collapse decimation with preserved topology boundaries (MeshLab). Geometries were remeshed to ensure uniformity (Instant Meshes) and the resulting 3D meshes exported (ABAQUS) for downstream application. The protocol has been applied successfully to fibroblast cytoskeletal reorganisation in the scleral connective tissue of the eye, under mechanical load that mimics internal eye pressure. While the method herein is specifically employed to reconstruct immunofluorescent confocal imaging data, it is also more widely applicable to other biological imaging modalities where accurate 3D cell structures are required.     

MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues

Background  

Comparative, functional, and developmental studies of animal morphology require accurate visualization of three-dimensional structures, but few widely applicable methods exist for non-destructive whole-volume imaging of animal tissues. Quantitative studies in particular require accurately aligned and calibrated volume images of animal structures. X-ray microtomography (microCT) has the potential to produce quantitative 3D images of small biological samples, but its widespread use for non-mineralized tissues has been limited by the low x-ray contrast of soft tissues. Although osmium staining and a few other techniques have been used for contrast enhancement, generally useful methods for microCT imaging for comparative morphology are still lacking.     

3D articulated growth model of the fetus skeleton,envelope and soft tissues

Fetal dosimetry studies require the development of accurate 3D models of the fetus. This paper proposes a 3D articulated fetal growth model including skeleton, body envelope, brain and lungs based on medical images of ten different fetuses acquired in clinical routine. The structures of interest were semi-manually segmented from the images and surface meshes were generated. A generic mesh of each structure has been deformed towards the segmented ones. By interpolating linearly between the subjects of the database, each structure can be estimated at any age and in any position. This process results in an automated model, the operator being only required to specify the age and position of the desired estimated fetus.     

Osteopenic mice: animal models of the aging skeleton

While our understanding of the developmental biology of the skeleton, like that of virtually every other subject in biology, has been transformed by recent advances in human and mouse genetics, we still know very little, in molecular and genetic terms, about skeletal physiology. Thus, among the many questions that are largely unexplained are the following: why is osteoporosis mainly a women's disease? How is bone mass maintained nearly constant between the end of puberty and the arrest of gonadal functions? Molecular genetics has emerged as a powerful tool to study previously unexplored aspects of the physiology of the skeleton. Among mammals, mice are the most promising animals for this experimental work. This has been previously demonstrated e.g. through the tremendous impact of the different osteopetrotic models on our molecular understanding of osteoclastic bone resorption. Until recently the only way of studying bone loss situations and osteoporosis in mice was by using ovariectomy with all its limitations. Today, however, we have access to more sophisticated osteoporotic mouse-models from four different origins: Transgenic mice (HSV-TK), knock-out mice (OPG), inbred-strains (SAMP6), and through physiological modulation (icv application). These new models have already taught us several important lessons. The first is, that bone remodeling is more than just an autocrine/paracrine process. Multiple experimental evidence has demonstrated that the latter regulation exists, but genetics prove that there is no functional cross-control between resorption and formation. The second lesson is, that remodeling is, at least in part, subject to central regulation. Thus, osteoporosis is partly a central or hypothalamic disease. However, the most dramatic change and the most important advantage we feel is, that today we have models to test a new hypothesis regarding the etiology of osteoporosis before it turns to dogma. Taken together, mouse-studies may lead to a shift in our physiological understanding of skeleton biology and to the emergence of novel paradigms. These, in turn, should help us to devise new treatments for degenerative diseases of the skeleton such as osteoporosis and its associated clinical problems.     

3D chromatin architecture and epigenetic regulation in cancer stem cells

Dedifferentiation of cell identity to a progenitor-like or stem cell-like state with increased cellular plasticity is frequently observed in cancer formation.During this process,a subpopulation of cells in tumours acquires a stem cell-like state partially resembling to naturally occurring pluripotent stem cells that are temporarily present during early embryogenesis.Such characteris-tics allow these cancer stem cells (CSCs) to give rise to the whole tumour with its entire cellular heterogeneity and thereby support metastases formation while being resistant to current cancer therapeutics.Cancer devel-opment and progression are demarcated by transcrip-tional dysregulation.In this article,we explore the epigenetic mechanisms shaping gene expression dur-ing tumorigenesis and cancer stem cell formation,with an emphasis on 3D chromatin architecture.Comparing the pluripotant stem cell state and epigenetic repro-gramming to dedifferentiation in cellular transformation provides intriguing insight to chromatin dynamics.We suggest that the 3D chromatin architecture could be used as a target for re-sensitizing cancer stem cells to therapeutics.     

Computer simulation modelling and visualization of 3D architecture of biological tissues

Recent technical improvements, such as 3D microscopy imaging, have shown the necessity of studying 3D biological tissue architecture during carcinogenesis. In the present paper a computer simulation model is developed allowing the visualization of the microscopic biological tissue architecture during the development of metaplastic and dysplastic lesions.The static part of the model allows the simulation of the normal, metaplastic and dysplastic architecture of an external epithelium. This model is associated to a knowledge base which contains only data on the nasal epithelium. The latter has been well studied by numerous authors and its lesional states are well known. An inference engine allows the initialization of the static model parameters. A statistical comparison between simulated epithelia and real epithelia is achieved by adjusting the parameter values during the simulation.The dynamic part of the model allows the simulation of a growth process on a 3D representation based on the static model. The main hypothesis is that nasal epithelium is submitted to a continuous transformation from normal to cancer through metaplasia and dysplasia. The evolution of each cell (represented by its nucleus) depends on its local environment and also on its heritage from its mother-cell.Simulation of tissue renewal of the nasal pseudostratified epithelium has been achieved. The evolution from normal to hyperplasia has been simulated. After modification of the cell cycle modelling, the simulation of the development of metaplastic foci has been obtained.     

Titin organisation and the 3D architecture of the vertebrate-striated muscle I-band

The giant muscle protein titin (connectin) is known to serve as a cytoskeletal element in muscle sarcomeres. It elastically restrains lengthening sarcomeres, it aids the integrity and central positioning of the A-band in the sarcomere and it may act as a template upon which some sarcomeric components are laid down during myogenesis. A puzzle has been how titin molecules, arranged systematically within the hexagonal A-band lattice of myosin filaments, can redistribute through the I-band to their anchoring sites in the tetragonal Z-band lattice. Recent work by Liversage and colleagues has suggested that there are six titin molecules per half myosin filament. Since there are two actin filaments per half myosin filament in a half sarcomere, this means that there are three titin molecules interacting with each Z-band unit cell containing one actin filament in the same sarcomere and one of opposite polarity from the next sarcomere. Liversage et al. suggested that the three titins might be distributed with two on an actin filament of one polarity and one on the filament of opposite polarity. Here, we build on this suggestion and discuss the transition of titin from the A-band to the Z-band. We show that there are good structural and mechanical reasons why titin might be organised as Liversage et al., suggested and we discuss the possible relationships between A-band arrangements in successive sarcomeres along a myofibril.     

3D analysis of chromosome architecture: advantages and limitations with SEM

Three-dimensional mitotic plant chromosome architecture can be investigated with the highest resolution with scanning electron microscopy compared to other microscopic techniques at present. Specific chromatin staining techniques making use of simultaneous detection of back-scattered electrons and secondary electrons have provided conclusive information on the distribution of DNA and protein in barley chromosomes through mitosis. Applied to investigate the structural effects of different preparative procedures, these techniques were the groundwork for the "dynamic matrix model" for chromosome condensation, which postulates an energy-dependent process of looping and bunching of chromatin coupled with attachment to a dynamic matrix of associated protein fibers. Data from SEM analysis shows basic higher order chromatin structures: chromomeres and matrix fibers. Visualization of nanogold-labeled phosphorylated histone H3 (ser10) with high resolution on chromomeres shows that functional modifications of chromatin can be located on structural elements in a 3D context.     

3D imaging of microstructure of spruce wood

Synchrotron radiation phase-contrast X-ray tomographic microscopy (srPCXTM) was applied to observation and identification of the features of spruce anatomy at the cellular lengthscale. The pilot experiments presented in the paper clearly revealed the features of the heartwood of Spruce (Picea abies [L.] Karst.), such as lumina and pits connecting the lumina, with a theoretical voxel size of 0.7 x 0.7 x 0.7 microm(3). The experiments were carried out on microspecimens of heartwood, measuring approximately 200 by 200 micrometers in cross-section. The technique for production and preparation of wood microsamples was developed within the framework of this investigation. The total porosity of the samples was derived and the values of the microstructural parameters, such as the diameters of tracheid, cell wall thicknesses and pit diameters were assessed non-invasively. Microstructural features as thin/small as approximately 1.5 microm were revealed and reconstructed in 3D. It is suggested that the position of sub-voxel-sized features (such as position of tori in the bordered pit pairs) can be determined indirectly using watershed segmentation. Moreover, the paper discusses the practical issues connected with a pipelined phase-contrast synchrotron-based microtomography experiment and the possible future potentials of this technique in the domain of wood science.     

3D and 4D imaging of immune cells in vitro and in vivo

Analyzing the dynamics of cellular immune responses, although performed for decades in immunologic research, has seen an enormous increase in the number of studies using this approach since the development of intravital 2-photon microscopy. Meanwhile, new insights into the dynamics of cellular immunity are being published on a daily basis. This review gives a short overview of the currently most widely used techniques, both on the microscopy side as well as on the experimental part. Difficulties and promises will be discussed. Finally, a personal selection of the most interesting findings of the first 6 years of intravital 2-photon microscopy for immunological questions will be given. The overall aim is to get the reader interested into this fascinating way of investigating the immune response by means of “dynamic histology”. This already has and will continue to broaden our view on how immune cells work in real life.