Episcopic Fluorescence Image Capture Research Articles

Three-Dimensional Imaging Analysis of the Developmental Process of Posterior Meniscofemoral Ligaments in Rat Embryos

Introduction: The posterior meniscofemoral ligament (pMFL) of knee joint is a ligament that runs posterior to the posterior cruciate ligament and it is known that the height of the pMFL attachment site causes meniscus avulsion. Therefore, understanding the three-dimensional (3D) structure of the pMFL attachment site is essential to better understand the pathogenesis of meniscus disorders. However, the developmental process of pMFL has not been well investigated. The purpose of this study was to analyze pMFL development in rat knee joints using 3D reconstructed images produced from episcopic fluorescence image capture (EFIC) images and examine its relationship with other knee joint components. Methods: Knee joints of Wistar rat embryos between embryonic day (E) 16 and E21 were observed with HE-stained tissues. Serial EFIC images of the hind limbs of E17–E21 were, respectively, captured from which 3D images were reconstructed and the features of pMFL structure: length and angle were measured. Besides, the chronological volume changes and the volume ratio of the knee joint components compared to E17 were calculated to identify the differences in growth by components. Results: pMFL was observed from E17 and was attached to the medial femoral condyle and lateral meniscus at all developmental stages, as in mature rats. The lack of marked variation in the attachment site and angle of the pMFL with the developmental stage indicates that the pMFL and surrounding knee joint components developed while maintaining their positional relationship from the onset of development. Conclusion: Current results may support to congenital etiology of meniscus disorder.

A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe CHD died before adulthood, with the maximum mortality during the first week of life. Remarkable advances in surgical techniques, diagnostic approaches, and medical management have led to marked improvements in outcomes. To address the critical research needs of understanding congenital heart defects, murine models have provided an ideal research platform, as they have very similar heart anatomy to humans and short gestation rates. The combination of genetic engineering with high-throughput phenotyping tools has allowed for the replication and diagnosis of structural heart defects to further elucidate the molecular pathways behind CHDs. The use of noninvasive fetal echocardiography to screen the cardiac phenotypes in mouse models coupled with the high fidelity of Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) histopathology with three-dimensional (3D) reconstructions enables a detailed view into the anatomy of various congenital heart defects. This protocol outlines a complete workflow of these methods to obtain an accurate diagnosis of murine congenital heart defects. Applying this phenotyping protocol to model organisms will allow for accurate CHD diagnosis, yielding insights into the mechanisms of CHD. Identifying the underlying mechanisms of CHD provide opportunities for potential therapies and interventions.

Novel Imaging Modalities for Human Embryology and Applications in Education.

Advances in imaging technology and development have recently enabled high-resolution three-dimensional (3D) imaging of embryos and fetuses. Embryos and fetuses stored at the Congenital Anomaly Research Center (Kyoto Collection of Human Embryos, Kyoto, Japan) were imaged using multiple modalities including magnetic resonance imaging, episcopic fluorescence image capture, and X-ray computed tomography, both in absorption and phase-contrasted configurations. Using the acquired images, 3D computer graphics were generated and a movie was created to gain further insight into understanding the developmental process. For educational purposes, self-learning materials were also produced. The present review article briefly discusses each project and the results of imaging studies performed using specimens from the Kyoto Collection of Human Embryos. Anat Rec, 301:1004-1011, 2018. © 2018 Wiley Periodicals, Inc.

Developing the Digital Kyoto Collection in Education and Research.

The Kyoto embryo collection was begun in 1961 by Dr. Hideo Nishimura. The collection has been continuously developed and currently contains over 44,000 human normal and abnormal specimens. Beginning online in 1997, the internet provided an opportunity to make embryos from the collection widely available for research and educational purposes (http://tiny.cc/Embryo). These embryonic development resources have been continuously published and available from that time until today. Published in Japanese as an Atlas of Embryonic Development. Published online as the Kyoto Human Embryo Visualization Project (http://atlas.cac.med.kyoto-u.ac.jp) and also as the Human Embryo Atlas (http://tiny.cc/Human_Embryo_Atlas). Published now electronically as a digital eBook (http://tiny.cc/Kyoto_Collection_eBook). This new digital format allows incorporation of whole embryo and histology manipulable images, labels, and a linked glossary. New imaging modalities of magnetic resonance imaging (MRI) and episcopic fluorescence image capture (EFIC) can also be easily displayed as animations. For research, the collection specimens and histological sections have been extensively studied and published in several hundred papers, discussed here and elsewhere in this special edition. I will also describe how the Kyoto collection will now form a major partner of a new international embryology research group, the Digital Embryology Consortium (https://human-embryology.org). The digital Kyoto collection will be made available for remote researcher access, analysis, and comparison with other collections allowing new research and educational applications. This work was presented at the 40th Anniversary Commemoration Symposium of the Congenital Anomaly Research Center, Graduate School of Medicine, Kyoto University, Japan, November, 2015. Anat Rec, 301:998-1003, 2018. © 2018 Wiley Periodicals, Inc.

Spatial Change of Cruciate Ligaments in Rat Embryo Knee Joint by Three-Dimensional Reconstruction.

This study aimed to analyze the spatial developmental changes of rat cruciate ligaments by three-dimensional (3D) reconstruction using episcopic fluorescence image capture (EFIC). Cruciate ligaments of Wister rat embryos between embryonic day (E) 16 and E20 were analyzed. Samples were sectioned and visualized using EFIC. 3D reconstructions were generated using Amira software. The length of the cruciate ligaments, distances between attachment points to femur and tibia, angles of the cruciate ligaments and the cross angle of the cruciate ligaments were measured. The shape of cruciate ligaments was clearly visible at E17. The lengths of the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) increased gradually from E17 to E19 and drastically at E20. Distances between attachment points to the femur and tibia gradually increased. The ACL angle and PCL angle gradually decreased. The cross angle of the cruciate ligaments changed in three planes. The primordium of the 3D structure of rat cruciate ligaments was constructed from the early stage, with the completion of the development of the structures occurring just before birth.

High‐resolution histological 3D‐imaging: Episcopic fluorescence image capture is widely applied for experimental animals

High‐resolution histological <scp>3D</scp>‐imaging: Episcopic fluorescence image capture is widely applied for experimental animals

Three-dimensional reconstruction of rat knee joint using episcopic fluorescence image capture

Development of the knee joint was morphologically investigated, and the process of cavitation was analyzed by using episcopic fluorescence image capture (EFIC) to create spatial and temporal three-dimensional (3D) reconstructions. Knee joints of Wister rat embryos between embryonic day (E)14 and E20 were investigated. Samples were sectioned and visualized using an EFIC. Then, two-dimensional image stacks were reconstructed using OsiriX software, and 3D reconstructions were generated using Amira software. Cavitations of the knee joint were constructed from five divided portions. Cavity formation initiated at multiple sites at E17; among them, the femoropatellar cavity (FPC) was the first. Cavitations of the medial side preceded those of the lateral side. Each cavity connected at E20 when cavitations around the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) were completed. Cavity formation initiated from six portions. In each portion, development proceeded asymmetrically. These results concerning anatomical development of the knee joint using EFIC contribute to a better understanding of the structural feature of the knee joint.

Imaging techniques for visualizing and phenotyping congenital heart defects in murine models

Mouse model is ideal for investigating the genetic and developmental etiology of congenital heart disease. However, cardiovascular phenotyping for the precise diagnosis of structural heart defects in mice remain challenging. With rapid advances in imaging techniques, there are now high throughput phenotyping tools available for the diagnosis of structural heart defects. In this review, we discuss the efficacy of four different imaging modalities for congenital heart disease diagnosis in fetal/neonatal mice, including noninvasive fetal echocardiography, micro-computed tomography (micro-CT), micro-magnetic resonance imaging (micro-MRI), and episcopic fluorescence image capture (EFIC) histopathology. The experience we have gained in the use of these imaging modalities in a large-scale mouse mutagenesis screen have validated their efficacy for congenital heart defect diagnosis in the tiny hearts of fetal and newborn mice. These cutting edge phenotyping tools will be invaluable for furthering our understanding of the developmental etiology of congenital heart disease.

Toward Routine Use of 3D Histopathology as a Research Tool

Three-dimensional (3D) reconstruction and examination of tissue at microscopic resolution have significant potential to enhance the study of both normal and disease processes, particularly those involving structural changes or those in which the spatial relationship of disease features is important. Although other methods exist for studying tissue in 3D, using conventional histopathological features has significant advantages because it allows for conventional histopathological staining and interpretation techniques. Until now, its use has not been routine in research because of the technical difficulty in constructing 3D tissue models. We describe a novel system for 3D histological reconstruction, integrating whole-slide imaging (virtual slides), image serving, registration, and visualization into one user-friendly package. It produces high-resolution 3D reconstructions with minimal user interaction and can be used in a histopathological laboratory without input from computing specialists. It uses a novel method for slice-to-slice image registration using automatic registration algorithms custom designed for both virtual slides and histopathological images. This system has been applied to >300 separate 3D volumes from eight different tissue types, using a total of 5500 virtual slides comprising 1.45 TB of primary image data. Qualitative and quantitative metrics for the accuracy of 3D reconstruction are provided, with measured registration accuracy approaching 120 μm for a 1-cm piece of tissue. Both 3D tissue volumes and generated 3D models are presented for four demonstrator cases.

Abstract 17475: High Throughput Screening and Diagnosis of Structural Heart Defects in Newborn Mice Using High Resolution Micro-Computed Tomography

Background: We are conducting a high-throughput chemical mutagenesis screen in mice to recover mutations causing congenital heart defects (CHD) using fetal echocardiography. Mutant fetuses identified to have cardiac defects by ultrasound screening must be further examined by time-consuming necropsy and histological analysis to confirm the CHD. We hypothesize post-mortem micro-CT may be used as a high throughput secondary screening tool for phenotyping CHD in fetal/newborn mice. Methods: Formalin fixed fetuses and neonatal mice were soaked with iodine for soft tissue visualization. Micro-CT scanning was carried out at 15 or 45μ m using the Siemens Inveon with a custom 12-pup cradle. Micro-CT diagnoses of suspected CHD were validated using episcopic fluorescence image capture (EFIC), a 3D high resolution histological imaging technique that provides registered image stacks suitable for digital resectioning in any imaging plane (Figure). Results. We micro-CT scanned 938 newborn mice and 58 late term fetuses and found a spectrum of CHD. This included ventricular septal defects (VSDs; 14.7%, n=146), endocardial cushion defects (0.9%, n=9), transposition of the great arteries (TGA; 1.2%, n=12), double outlet right ventricle (DORV; 2.2%, n=22), pulmonary atresia (PA; 0.1%, n=1), tricuspid atresia (0.5%, n=5), right aortic arch (RAA; 0.3%, n=3), interrupted aortic arch (0.2%; n=2), and dextrocardia (0.5%; n=5). Subsequent necropsy/EFIC imaging of 45 pups confirmed all RAA diagnoses (100%; n=3), 91% of the VSD (n=29/32), 90% of the TGA/DORV/PA (n=9/10) and 100% of the dextrocardia (n=5/5). We had false positive rate of 6% (n=2) for VSD and 10% (n=1) for TGA/DORV, while a false negative rate of 3% (n=1) was observed for VSD. Conclusions: Our studies showed micro-CT has a low false negative CHD detection rate, thus making it a valuable secondary imaging tool for enhancing the diagnosis of structural heart defects in large-scale mutagenesis screens.

High Throughput Congenital Heart Disease Phenotyping in Mice with Noninvasive Fetal Echocardiography

Fetal ultrasound imaging is extensively used clinically for congenital heart disease (CHD) diagnosis. We previously showed fetal echocardiography using the Acuson Sequoia clinical ultrasound system with a 15 MHz transducer was highly effective in recovering mutants with CHD in a mouse ethylnitrosourea (ENU) mutagenesis screen. Building on this study, we initiated a new mouse ENU screen for mutations causing CHD, with the goal of scanning 100,000 mouse fetuses for 5X genome coverage. ENU mutagenized mice are bred in a G2XG1 backcross breeding scheme to generate G3 fetuses for ultrasound scanning at E14.5–15.5. Litters with Acuson findings are secondarily scanned with the higher resolution Vevo2100 ultrasound system using a 30 MHz transducer. To confirm the CHD diagnosis, fetuses/newborn pups are further analyzed using a combination of microCT, necropsy and/or histopathology with episcopic fluorescence image capture. Thus far, ultrasound scanning showed 4% of the G3 fetuses (n=3099) from 60% of the G1 pedigrees (n=89) exhibit cardiac defects, a similar yield to our previous screen. The most common cardiac defects include atrial/ventricular septal defects, outflow tract alignment defects, and hypertrophy. This new forward genetic screen promises to be highly productive in yielding new insights into the genetic etiology of CHD. Funded by U01HL098180

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Stem cells, from the ground up (Caenorhabditis elegans as a Model for Stem Cell Biology by Pradeep M. Joshi, Misty R. Riddle, Nareg J.V. Djabrayan, and Joel H. Rothman, Dev Dyn 239:1539–1554) C. elegans, may not be the first model system that comes to mind when considering questions of stem cell biology. The organism's only true stem cells are the germline, and it cannot be maintained in cell culture. Nevertheless the amenity of C. elegans to genetic, cell, and molecular manipulations have allowed scientists to use innovative approaches to milk additional information from other cell populations with stem-cell like properties. Epidermal seam cells balance proliferation with differentiation, embryonic progenitor cells progress from a state of pluripotency to cell fate commitment, and the “Y” rectal epithelial cell naturally transforms into a neuron during larval development. This review illustrates how the different cell populations have illuminated mechanisms behind stem cell-niche interactions, self-renewal, cell fate restriction, and transdifferentiation. Although discovered in the lowly worm, many of these themes are proving to be conserved in mammalian systems. Developing a human embryo atlas (Developmental Atlas of the Early First Trimester Human Embryo by Shigehito Yamada, Rajeev R. Samtani, Elaine S. Lee, Elizabeth Lockett, Chigako Uwabe, Kohei Shiota, Stasia A. Anderson, and Cecilia W. Lo, Dev Dyn 239:1585–1595) Many believe that the goal of developmental biology research is to inform human health. However, because our knowledge of human embryogenesis is based largely on drawings and wax models, it lags far behind that of popular model systems, making accurate comparisons impossible. Here, Yamada and colleagues present a much-needed, updated Human Embryo Atlas (http://apps.devbio.pitt.edu/humanatlas/; login: “Human”, password: “Embryo”) encompassing most of the first trimester, when development of major organs and tissues take place. The high-resolution images captured by 21st century techniques—magnetic resonance (MR) imaging and episcopic fluorescence image capture (EFIC)—can be virtually sectioned from any angle, and used to make three-dimensional (3D) reconstructions. The versatile images will help identify potential differences between development of humans and the model organisms that have so far informed our current understanding of vertebrate development, and will facilitate clinical assessments of embryonic health. dalmatian's new trick (Staying Alive: Dalmatian Mediated Blocking of Apoptosis Is Essential for Tissue Maintenance by Bilal E. Kerman and Deborah J. Andrew, Dev Dyn 239:1609–1621) The novel Drosophila nuclear protein Dalmatian (Dmt) was already known for one trick: regulating mitotic spindle assembly. Here, Kerman and Andrew show that dmt also keeps specific cell types from playing dead. In dmt mutants, trachea and salivary gland tubulogenesis is spotty—with networks displaying frequent gaps and breaks. The mutants also show increased expression of the apoptotic activators reaper (rpr) and head involution defective (hid), and the trachea displays enhanced apoptosis during latter stages of morphogenesis (st. 12–14). Dmt salivary gland, and trachea defects are rescued in the background of a deficiency that removes apoptotic inhibitors, and by tissue-specific expression of an anti-apoptotic protein, p35. Of interest, st. 12–14 dmt mutants are sensitized to radiation-induced apoptosis. Because Dmt localizes to heterochromatin, the authors speculate that the protein participates in known epigenetic silencing of rpr and hid during these stages. Dmt's role repressing apoptotic genes probably occurs after cells have ceased dividing, and thus is independent from it's role in mitotic spindle assembly. Always eager to please, Dmt can perform two different tricks.

Mouse Models of Congenital Heart Defects

One of the puzzling things about genetic disruptions in mice that affect heart development is the overwhelming number of conotruncal anomalies and the infrequency with which certain other malformations are reported. For instance, venous malformations such as total anomalous pulmonary venous return (TAPVR) or partial anomalous pulmonary venous return (PAPVR) have not been reported in a mutant mouse. Coronary artery anomalies are reported infrequently, and when they are reported it is because they are obvious. Conotruncal anomalies such as common trunk, double-outlet right ventricle, and pulmonary stenosis with overriding aorta are frequently associated with mouse germ line and conditional gene deletions. Although conotruncal malformations represent the highest percentage of congenital heart defects in the human population, TAPVR and PAPVR occur in a significant number of babies. Further, coronary anomalies affecting the stems of the coronary arteries frequently are seen in conotruncal malformations in babies but are rarely reported in mouse models. Article see p 314 Isolated TAPVR is a rare lesion and occurs in only about 1 in 17 000 live births.1 PAPVR is much more common and has been reported in 1 in 160 individuals (0.6%) and 0.4% to 0.7% of postmortem examinations, with 90% of these associated with an atrial septal defect.2 The majority of patients with TAPVR have symptoms of cyanosis and congestion within the first year of life. If left untreated, about 80% would die. In contrast, PAPVR frequently is not discovered until adulthood and is not usually a cause of death. Although the overall incidence of PAPVR in autopsy cases has been estimated to be 0.7%,3 the true prevalence may be higher,4 as several reports have appeared with PAPVR as an incidental finding that in most of these cases is asymptomatic. PAPVR can present as an isolated structural abnormality, but it …

Ventricular Rotation Is Independent of Cardiac Looping: A Study in Mice With Situs Inversus Totalis Using Speckle-Tracking Echocardiography

The authors conducted an ultrasound interrogation of a mutant mouse model with a Dnah5 mutation to determine whether cardiac mechanics may be affected by reversal of cardiac situs. This mutant is a bona fide model of primary ciliary dyskinesia, with surviving homozygous mice showing either situs solitus (SS) or situs inversus totalis (SI). High-frequency ultrasound interrogations of 27 neonatal and infant Dnah5 mutant mice, 16 with SS and 11 with SI, were conducted using an ultra-high-frequency biomicroscope. Electrocardiographic and respiratory gating were used to reconstruct high-resolution two-dimensional cines at 1,000 Hz, with speckle-tracking echocardiography used to further analyze midchamber and apical rotation. All SS mice exhibited the expected counterclockwise apical rotation as viewed caudocranially, and surprisingly, the same counterclockwise motion was also observed in SI mice. Speckle-tracking analysis confirmed counterclockwise systolic rotation in both SS and SI mice, and this increased in magnitude from the subepicardium to the endocardium and from the papillary muscles to the apex. The magnitude of apical endocardial rotation was not different for SS and SI mice (5.64+/-0.75 degrees and 5.76+/-1.90 degrees, respectively, P=.93). The anatomic segments responsible for the largest components of apical endocardial systolic rotation differed between the SS and SI hearts (P=.004). In both, the two largest contributors to rotation were offset 180 degrees from each other, but the anatomic regions differed between them. In SS hearts, maximal regional rotation occurred at the anterior mid-septum and posterolateral free wall, while in SI hearts, it was derived from the posterior septum and the anterolateral free wall. Analysis by episcopic fluorescence image capture histology of representative SI and SS mice showed normal intracardiac and segmental anatomy ({S,D,S} or {I,L,I}) without intracardiac defects. These results show that mirror-image cardiac looping did not result in mirror-image rotation of the morphologic left ventricle. These findings suggest that further studies are warranted to evaluate whether fiber orientation and cardiac mechanics may be abnormal in individuals with reversal of cardiac situs. The results of this study indicate that cardiac looping and myofiber orientation may be independently regulated.

Digital Atlas of Human Embryonic Development by High Resolution Imaging

Embryos undergo complicated morphogenetic changes during organogenesis. Detailed characterization of the remodeling will help in elucidating the etiology of the congenital anomalies. The analysis of such changes require the visualization of embryonic structures in three‐dimensions (3D). To generate high resolution 3D images through the period of organogenesis, human embryos from the Kyoto Collection at Carnegie stages 13 to 23 were imaged by magnetic resonance imaging (MRI) and with episcopic fluorescence image capture (EFIC) ‐ a novel high resolution histological technique that provides perfectly registered serial 2D images suitable for high fidelity 3D rendering. Embryos were first MRI scanned using a 7T super‐conducting magnet, followed by EFIC imaging. Obtained high‐resolution images were resectioned digitally in multiple planes, allowing detailed visualization of anatomic structures in the developing embryo. Using these data sets, we constructed a digital atlas, with each stage represented by serial 2D image stacks shown in three orthogonal planes. The Atlas also includes annotations delineating important anatomic structures and developmental milestones, as well as 3D Quicktime movies in different perspectives. This Atlas of the Human Embryo should serve as an invaluable resource for morphologic information of human development, and for studying the etiology of the congenital anomalies.

Human breast duct anatomy, the ‘sick lobe’ hypothesis and intraductal approaches to breast cancer

Information about central and peripheral duct anatomy is a requirement for developing intraductal approaches to human breast cancer, but remains sparse. This study looks at the acquisition and digital modelling of data describing breast duct branching from thick ('subgross') sections using data structures from the neurosciences, and at high-throughput imaging of duct anatomy in the nipple. The branching of a large breast duct system was modelled using data extracted from cleared and stained 2 mm 'subgross' sections of an autopsy breast using a public-domain neuron modelling program (CVAPP), and episcopic fluorescence image capture (EFIC) was used to collect a stack of 1100 autofluorescence images of ducts in a mastectomy nipple. The duct skeleton was captured in 440 line segments with some pruning of terminal ducts. Extracting this data manually in a usable form was, however, laborious and error prone, emphasising the need for improved morphological informatics. EFIC captured anatomical detail and subsequent 3D reconstruction was consistent with the distinction between 'type A' and 'type B' nipple ducts proposed by Going and Moffat (J Pathol 203: 538-544, 2004). Whole-lobe duct modelling and EFIC reveal central and peripheral duct anatomy in human breast. Such knowledge is required for understanding normal breast development, the growth of cancer precursors, and for developing the intraductal approach to breast cancer.

Rapid high resolution three dimensional reconstruction of embryos with episcopic fluorescence image capture

One of the overarching goals in developmental biology is the elucidation of mechanisms that elaborate form and function. To this end, an accurate morphological description of embryonic development is essential. However, visualizing dynamic changes in the three-dimensional (3D) structure of the developing embryo has been a "holy grail" in the field of developmental biology. The fundamental difficulties that have hindered all efforts in 3D reconstruction using two-dimensional (2D) image stacks revolve around the seemingly intractable problems of section registration and distortion. A remarkably simple solution has come about with the development of a new technique referred to as episcopic fluorescence image capture (EFIC). With EFIC imaging, tissue autofluorescence is used to image the block face prior to cutting each section. The 2D resolution obtained is close to that achieved by histology, and such 2D image stacks can be readily reconstructed in 3D. The 3D models generated provide fine structural details with resolution unmatched by 3D reconstructions obtained with any other imaging modalities. Given the perfect registration of EFIC image stacks, another important capability provided by EFIC is digital resectioning in any plane. This provides complete flexibility in the selection of optimal virtual sectioning planes for viewing different features in a specimen, and is invaluable for analyzing dynamic changes in tissue structure in the developing embryo. The capabilities provided by EFIC for rapid high resolution 3D reconstruction together with digital resectioning make this an unparalleled tool for characterizing morphogenetic events in the developing embryo. Although our review is focused on using EFIC for studying embryonic development, it is important to note that there is no intrinsic limitation on the size of the specimen that can be analyzed by EFIC imaging. Overall, EFIC should serve as an important imaging technique that will complement other 3D imaging modalities such as MRI and optical tomography. Given the feasibility of generating EFIC image stacks using cryoembedded or polyethylene glycol (PEG)-embedded specimens, there is the possibility that EFIC may be combined with 3D RNA or protein expression profiling. Together, such studies may help further elucidate the relationship between form and function.