Tissue arrays as fiducial markers for section alignment in 3-D reconstruction technology

Combination of conventional histology and the three-dimensional spatial view of tissue structures offers new prospects for understanding and diagnosing nature and development of human diseases. The essential technical problem related to three-dimensional reconstruction in histopathology is represented by the correct alignment of serial sections. During the past years several methods have been proposed but failed to become popular because of their limits in terms of time consume and restricted applicability. We aimed to overcome this problem by applying the technology of Tissue Array, thus by positioning adequate fiducial markers from specific "donor" blocks into the "recipient" paraffin block of interest. Digitized pictures of serially cut sections were aligned according to the tissue markers embedded by Tissue Array, and then processed with specific softwares for three-dimensional reconstruction. Thirteen models, including fetal hearts, breast and thyroid carcinomas, were elaborated. We found the procedure to be easy, fast and reproducible. Moreover, by selectively embedding the fiducial markers according to specific angles, the Tissue Arrays can be exploited in order to establish the distance between sections. This original methodology of incorporating Tissue Arrays into paraffin blocks as fiducial markers for three-dimensional reconstruction has a potential impact on histology for research purposes and diagnostic applications.     

Accuracy of computer-aided geometric 3D reconstruction based on histological serial microgrinding preparation

Purpose: For our research on computer-optimised and automated cochlear implant surgery, we pursue a model-based approach to overcome the limitations of currently available clinical imaging modalities. A serial cross section preparation procedure has been developed and evaluated concerning accuracy to serve for modelling of a digital anatomic atlas to make delicate soft tissue structures available for pre-operative planning.

Methods: A special grinding tool was developed allowing the setting of a specific amount of abrasion as equidistant slice thickness was considered a crucial step. Additionally, each actual abrasion was accurately measured and used during three-dimensional reconstruction of the serial cross-sectional images obtained via digital photo documentation after each microgrinding step. A well-known reference object was prepared using this procedure and evaluated in terms of accuracy.

Results: Reconstruction of the whole sample was achieved with an error less than 0.4%, and the edge lengths in the direction of abrasion could be reconstructed with an average error of 0.6 ± 0.3 mm; both prove the realisation of equidistant abrasion. Using artificial registration fiducials and a custom-made algorithm for image alignment, parallelism and rectangularity could be preserved with average errors less than 0.4° ± 0.3°.

Conclusion: We present a systematic, practicable and reliable method for the geometrically accurate reconstruction of anatomical structures, which is especially suitable for the middle and inner ear anatomy including soft tissue structures. For the first time, the quality of such a reconstruction process has been quantified and successfully proven for its usability.     

A biplanar reconstruction method based on 2D and 3D contours: application to the distal femur

A three-dimensional (3D) reconstruction algorithm based on contours identification from biplanar radiographs is presented. It requires, as technical prerequisites, a method to calibrate the biplanar radiographic environment and a surface generic object (anatomic atlas model) representing the structure to be reconstructed. The reconstruction steps consist of: the definition of anatomical regions, the identification of 2D contours associated to these regions, the calculation of 3D contours and projection onto the radiographs, the associations between points of the X-rays contours and points of the projected 3D contours, the optimization of the initial solution and the optimized object deformation to minimize the distance between X-rays contours and projected 3D contours. The evaluation was performed on 8 distal femurs comparing the 3D models obtained to CT-scan reconstructions. Mean error for each distal femur was 1 mm.     

A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography

We describe a novel approach for the accurate alignment of images in electron tomography of vitreous cryo-sections. Quantum dots, suspended in organic solvents at cryo-temperatures, are applied directly onto the sections and are subsequently used as fiducial markers to align the tilt series. Data collection can be performed from different regions of the vitreous sections, even when the sections touch the grid only at a few places. We present high-resolution tomograms of some organelles in cryo-sections of human skin cells using this method. The average error in image alignment was about 1nm and the resolution was estimated to be 5-7nm. Thus, the use of section-attached quantum dots as fiducial markers in electron tomography of vitreous cryo-sections facilitates high-resolution in situ 3D imaging of organelles and macromolecular complexes in their native hydrated state.     

A fast method for implicit surface reconstruction based on radial basis functions network from 3D scattered points

A method for arbitrary surface reconstruction from 3D large scattered points is proposed in this paper. According to the properties of 3D points, e.g. the non-uniform distribution and unknown topology, an implicit surface model is adopted based on radial basis functions network. And because of the property of locality of radial basis function, the method is fast and robust in surface reconstruction. Furthermore, an adapted K-Means algorithm is used to reduce reconstruction centers. For features completeness, two effective methods are introduced to extract the feature points before the adapted K-Means algorithm. Experiment results show that the presented approach is a good solution for reconstruction from 3D large scattered points.     

A novel 3D mouse embryo atlas based on micro-CT

The goal of the International Mouse Phenotyping Consortium (IMPC) is to phenotype targeted knockout mouse strains throughout the whole mouse genome (23,000 genes) by 2021. A significant percentage of the generated mice will be embryonic lethal; therefore, phenotyping methods tuned to the mouse embryo are needed. Methods that are robust, quantitative, automated and high-throughput are attractive owing to the numbers of mice involved. Three-dimensional (3D) imaging is a useful method for characterizing morphological phenotypes. However, tools to automatically quantify morphological information of mouse embryos from 3D imaging have not been fully developed. We present a representative mouse embryo average 3D atlas comprising micro-CT images of 35 individual C57BL/6J mouse embryos at 15.5 days post-coitum. The 35 micro-CT images were registered into a consensus average image with our automated image registration software and 48 anatomical structures were segmented manually. We report the mean and variation in volumes for each of the 48 segmented structures. Mouse organ volumes vary by 2.6-4.2% on a linear scale when normalized to whole body volume. A power analysis of the volume data reports that a 9-14% volume difference can be detected between two classes of mice with sample sizes of eight. This resource will be crucial in establishing baseline anatomical phenotypic measurements for the assessment of mutant mouse phenotypes, as any future mutant embryo image can be registered to the atlas and subsequent organ volumes calculated automatically.     

A novel method for somatic cell nuclear transfer to mouse embryonic stem cells

Nuclear reprogramming by somatic cell nuclear transfer (SCNT) provides a practical approach for generating autologous pluripotent cells from adult somatic cells. It has been shown that murine somatic cells can also be reprogrammed to a pluripotent-like state by fusion with embryonic stem (ES) cells. Typically, the first step in SCNT involves enucleation of the recipient cell. However, recent evidence suggests that enucleated diploid ES cells may lack reprogramming capabilities. Here we have developed methods whereby larger tetraploid ES cells are first generated by fusion of two mouse ES cell lines transfected with plasmids carrying different antibiotic-resistance cassettes, followed by double antibiotic selection. Tetraploid ES cells grown on tissue culture disks or wells can be efficiently enucleated (up to 99%) using a combination of cytochalasin B treatment and centrifugation, with cytoplasts generated from these cells larger than those obtained from normal diploid ES cells. Also, we show that the enucleation rate is dependent on centrifugation time and cell ploidy. Further, we demonstrate that normal diploid ES cells can be fused to tetraploid ES cells to form heterokaryons, and that selective differential centrifugation conditions can be applied where the tetraploid nucleus is removed while the diploid donor nucleus is retained. This technology opens new avenues for generating autologous, diploid pluripotent cells, and provides a dynamic model for studying nuclear reprogramming in ES cells.     

A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development

Primary mouse hepatocytes are an important tool in the biomedical research field for the assessment of hepatocyte function. Several methods for hepatocyte isolation have been published; however, many of these methods require extensive handling and can therefore compromise the viability and function of the isolated cells. Since one advantage of utilizing freshly isolated cells is to maintain an environment in which the cells are more comparable to their in vivo state, it is important to have robust methods that produce cells with high viability, good purity and that function in a similar manner to that in their in vivo state. Here we describe a modified two-step method for the rapid isolation and characterization of mouse primary hepatocytes that results in high yields of viable cells. The asialoglycoprotein receptor (ASGPR), which is one of the most abundant cell surface receptors on hepatocytes, was used to monitor the function of the isolated hepatocytes by demonstrating specific binding of its ligand using a newly developed flow cytometry based ligand-receptor binding assay. Also, an in vitro screening method for siRNA drug candidates was successfully developed utilizing freshly isolated hepatocytes with minimum culture time.     

Ribostral: an RNA 3D alignment analyzer and viewer based on basepair isostericities

RNA atomic resolution structures have revealed the existance of different families of basepair interactions, each of which with its own isosteric sub-families. Ribostral (Ribonucleic Structural Aligner) is a user-friendly framework for analyzing, evaluating and viewing RNA sequence alignments with at least one available atomic resolution structure. It is the first of its kind that makes direct and easy- to-understand superposition of the isostericity matrices of basepairs observed in the structure onto sequence alignments, easily indicating allowed and unallowed substitutions at each BP position. Potential mistakes in the alignments can then be corrected using other sequence editing software. Ribostral has been developed and tested under Windows XP, and is capable of running on any PC or MAC platform with MATLAB 7.1 (SP3) or higher installed version. A stand-alone version is also available for the PC platform. AVAILABILITY: http://rna.bgsu.edu/ribostral.     

Automatic system for 3D reconstruction of the chick eye based on digital photographs

The geometry of anatomical specimens is very complex and accurate 3D reconstruction is important for morphological studies, finite element analysis (FEA) and rapid prototyping. Although magnetic resonance imaging, computed tomography and laser scanners can be used for reconstructing biological structures, the cost of the equipment is fairly high and specialised technicians are required to operate the equipment, making such approaches limiting in terms of accessibility. In this paper, a novel automatic system for 3D surface reconstruction of the chick eye from digital photographs of a serially sectioned specimen is presented as a potential cost-effective and practical alternative. The system is designed to allow for automatic detection of the external surface of the chick eye. Automatic alignment of the photographs is performed using a combination of coloured markers and an algorithm based on complex phase order likelihood that is robust to noise and illumination variations. Automatic segmentation of the external boundaries of the eye from the aligned photographs is performed using a novel level-set segmentation approach based on a complex phase order energy functional. The extracted boundaries are sampled to construct a 3D point cloud, and a combination of Delaunay triangulation and subdivision surfaces is employed to construct the final triangular mesh. Experimental results using digital photographs of the chick eye show that the proposed system is capable of producing accurate 3D reconstructions of the external surface of the eye. The 3D model geometry is similar to a real chick eye and could be used for morphological studies and FEA.     

A computer vision based method for 3D posture estimation of symmetrical lifting

Work-related musculoskeletal disorders (WMSD) are commonly observed among the workers involved in material handling tasks such as lifting. To improve work place safety, it is necessary to assess musculoskeletal and biomechanical risk exposures associated with these tasks. Such an assessment has been mainly conducted using surface marker-based methods, which is time consuming and tedious. During the past decade, computer vision based pose estimation techniques have gained an increasing interest and may be a viable alternative for surface marker-based human movement analysis. The aim of this study is to develop and validate a computer vision based marker-less motion capture method to assess 3D joint kinematics of lifting tasks. Twelve subjects performing three types of symmetrical lifting tasks were filmed from two views using optical cameras. The joints kinematics were calculated by the proposed computer vision based motion capture method as well as a surface marker-based motion capture method. The joint kinematics estimated from the computer vision based method were practically comparable to the joint kinematics obtained by the surface marker-based method. The mean and standard deviation of the difference between the joint angles estimated by the computer vision based method and these obtained by the surface marker-based method was 2.31 ± 4.00°. One potential application of the proposed computer vision based marker-less method is to noninvasively assess 3D joint kinematics of industrial tasks such as lifting.     

A rapid population method for action spectra applied to Halobacterium halobium.

We have developed a simple and rapid technique for measuring the action spectra for phototaxis of populations of microorganisms and applied it to halobacteria. A microscope with a dark-field condenser was used to illuminate the cell suspension in a sealed chamber with light of wavelength greater than 750 nm; in this region of the spectrum, the halobacteria show no phototactic response. A 150-micron spot of light from a xenon arc lamp, whose wavelength and intensity can be varied, was projected through the objective lens into the center of the dark field. The objective lens imaged this measuring spot through a 780-nm cut-off filter on an aperture in front of a photomultiplier. The intensity of the scattered 750-nm light, and therefore the photomultiplier current, is proportional to the number of cells in the measuring spot. A third lamp provided background light of variable wavelength and intensity through the dark-field condenser. To minimize secondary effects due to large changes in cell density, we recorded the initial changes in the photomultiplier current over 1 min after the actinic light had been switched on. By plotting the rate of change against wavelength, we obtained action spectra after the proper corrections for changes in light intensity with wavelength were applied and saturation effects were avoided.     

A reliable and economical method for gaining mouse embryonic fibroblasts capable of preparing feeder layers

Mouse embryonic fibroblasts (MEFs) are widely used to prepare feeder layers for culturing embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in vitro. Transportation lesions and exorbitant prices make the commercially obtained MEFs unsuitable for long term research. The aim of present study is to establish a method, which enables researchers to gain MEFs from mice and establish feeder layers by themselves in ordinary laboratories. MEFs were isolated from ICR mouse embryos at 12.5–17.5 day post-coitum (DPC) and cultured in vitro. At P2–P7, the cells were inactivated with mitomycin C or by X-ray irradiation. Then they were used to prepare feeder layers. The key factors of the whole protocol were analyzed to determine the optimal conditions for the method. The results revealed MEFs isolated at 12.5–13.5 DPC, and cultured to P3 were the best choice for feeder preparation, those P2 and P4–P5 MEFs were also suitable for the purpose. The P3–P5 MEFs treated with 10 μg/ml of mitomycin C for 3 h, or irradiated with X-ray at 1.5 Gy/min for 25 Gy were the most suitable feeder cells. Treating MEFs with 10 μg/ml of mitomycin C for 2.5 h, 15 μg/ml for 2.0 h, or irradiating the cells with 20 Gy of X-ray at 2.0 Gy/min could all serve as alternative methods for P3–P4 cells. Our study provides a reliable and economical way to obtain large amount of qualified MEFs for long term research of ESCs or iPSCs.     

Dynamic simulation of leaf area index for the soybean canopy based on 3D reconstruction

Leaf area index (LAI) of the soybean canopy was an important indicator to reflect the growth and development of the soybean plant. However, the traditional LAI measurement in the field was expensive, time-consuming, and challenging to achieve high accuracy. Thus, in this study, a calculation method of LAI for the soybean canopy based on 3D reconstruction was proposed, and the dynamic simulation model of canopy LAI was established. First, northeast soybean varieties of Kangxianchong8 and Dongnong252 were taken as the research objects, and a multi-source image synchronous acquisition platform for soybean canopy based on Kinect 2.0 was constructed to obtain the canopy image data from the V3 to R7 growth periods. Second, the 3D structure of the soybean canopy was reconstructed by conditional filtering and statistical filtering. Third, a soybean LAI estimation method was established by canopy analysis. The determination coefficient R² between the calculated value and the standard value of LAI was greater than 0.99. Finally, a dynamic simulation model of soybean LAI was established based on the Richards model and the genetic parameters of varieties. The results showed that the accuracy of the dynamic simulation model reached above 0.99, which realized the critical technology of rapid detection and dynamic simulation of LAI for the soybean canopy, and provided quantitative dynamic prediction and technical support for scientific regulation of ecology and morphology for the soybean canopy.     

A simplified method to generate serotonergic neurons from mouse embryonic stem and induced pluripotent stem cells

We have developed a new simple method to induce serotonergic neurons from embryonic stem (ES) and induced pluripotent stem cells. When ES or induced pluripotent stem cells were cultured on a thick gel layer of Matrigel, most colonies extended TuJ1-positive neurites. We found that noggin, a known antagonist of bone morphogenic protein, induces ES cells to express genes involved in serotonergic differentiation, such as Nkx2.2, Pet-1, Sonic hedgehog, tryptophan hydroxylase 2, and serotonin transporter, as well as increases high potassium-induced release of serotonin. To concentrate serotonergic neurons, ES cells carrying Pet-1-enhancer-driven enhanced green fluorescent protein were differentiated and sorted into about 80% pure cultures of serotonergic neurons. Whole cell voltage-clamp recordings showed a voltage-dependent current in dissociated neurons. This simplified method provides an alternative option for serotonergic differentiation of pluripotent stem cells and will likely contribute a deeper understanding regarding the nature of serotonergic neurons and open new therapeutic perspectives for the treatment of psychiatric disorders.     

A collaborative framework for 3D alignment and classification of heterogeneous subvolumes in cryo-electron tomography

The limitation of using low electron doses in non-destructive cryo-electron tomography of biological specimens can be partially offset via averaging of aligned and structurally homogeneous subsets present in tomograms. This type of sub-volume averaging is especially challenging when multiple species are present. Here, we tackle the problem of conformational separation and alignment with a “collaborative” approach designed to reduce the effect of the “curse of dimensionality” encountered in standard pair-wise comparisons. Our new approach is based on using the nuclear norm as a collaborative similarity measure for alignment of sub-volumes, and by exploiting the presence of symmetry early in the processing. We provide a strict validation of this method by analyzing mixtures of intact simian immunodeficiency viruses SIV mac239 and SIV CP-MAC. Electron microscopic images of these two virus preparations are indistinguishable except for subtle differences in conformation of the envelope glycoproteins displayed on the surface of each virus particle. By using the nuclear norm-based, collaborative alignment method presented here, we demonstrate that the genetic identity of each virus particle present in the mixture can be assigned based solely on the structural information derived from single envelope glycoproteins displayed on the virus surface.     

JULIDE: a software tool for 3D reconstruction and statistical analysis of autoradiographic mouse brain sections

In this article we introduce JULIDE, a software toolkit developed to perform the 3D reconstruction, intensity normalization, volume standardization by 3D image registration and voxel-wise statistical analysis of autoradiographs of mouse brain sections. This software tool has been developed in the open-source ITK software framework and is freely available under a GPL license. The article presents the complete image processing chain from raw data acquisition to 3D statistical group analysis. Results of the group comparison in the context of a study on spatial learning are shown as an illustration of the data that can be obtained with this tool.     

Digital stereophotogrammetry based on circular markers and zooming cameras: evaluation of a method for 3D analysis of small motions in orthopaedic research

Background  

Orthopaedic research projects focusing on small displacements in a small measurement volume require a radiation free, three dimensional motion analysis system. A stereophotogrammetrical motion analysis system can track wireless, small, light-weight markers attached to the objects. Thereby the disturbance of the measured objects through the marker tracking can be kept at minimum. The purpose of this study was to develop and evaluate a non-position fixed compact motion analysis system configured for a small measurement volume and able to zoom while tracking small round flat markers in respect to a fiducial marker which was used for the camera pose estimation.