Magnetic Resonance Imaging Microscopy Research Articles

Automated segmentation of the actively stained mouse brain using multi-spectral MR microscopy

Magnetic resonance microscopy (MRM) has created new approaches for high-throughput morphological phenotyping of mouse models of diseases. Transgenic and knockout mice serve as a test bed for validating hypotheses that link genotype to the phenotype of diseases, as well as developing and tracking treatments. We describe here a Markov random fields based segmentation of the actively stained mouse brain, as a prerequisite for morphological phenotyping. Active staining achieves higher signal to noise ratio (SNR) thereby enabling higher resolution imaging per unit time than obtained in previous formalin-fixed mouse brain studies. The segmentation algorithm was trained on isotropic 43-μm T1- and T2-weighted MRM images. The mouse brain was segmented into 33 structures, including the hippocampus, amygdala, hypothalamus, thalamus, as well as fiber tracts and ventricles. Probabilistic information used in the segmentation consisted of (a) intensity distributions in the T1- and T2-weighted data, (b) location, and (c) contextual priors for incorporating spatial information. Validation using standard morphometric indices showed excellent consistency between automatically and manually segmented data. The algorithm has been tested on the widely used C57BL/6J strain, as well as on a selection of six recombinant inbred BXD strains, chosen especially for their largely variant hippocampus.

Highlights in DD

“Highlights” is a new feature that 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 will spotlight 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. Shapely models (Dev Dyn 2006;235:2907–2919) In classic experiments, Spemann and Mangold transplanted dorsal “organizer” cells from one newt to another, creating the unforgettable two-headed embryo. This and later works led to the hypothesis that the Spemann organizer specifies the primary body axes. In this review, Meinhardt eschews convention and proposes that the Spemann organizer plays a secondary role in axis formation. His model states that the head anterior/posterior (AP) axis is specified by the blastopore, and the trunk's by oscillating expression of sequentially posteriorizing genes. The role of the Spemann organizer is to trigger formation of head and trunk midline organizers (prechordal plate and notochord), which specify the dorsal/ventral (DV) axis. Unlike other models, Meinhardt's predicts the correct AP and DV orientation of an induced second axis. This provocative model will surely shape the interpretation of results from both new and old experiments in the field. Creating boundaries (Dev Dyn 2006;235:3051–3058) Like a border fence between countries, cells with distinct fates need boundaries to keep one population from invading the other. Major and Irvine show for the first time that, in the Drosophila wing disc, Myosin II is a necessary boundary component downstream of Notch signaling. Of interest, Myosin II is required for DV, but not AP compartmentalization, showing that not all boundaries are alike. Building on their previous work, the authors propose that Myosin II creates a contractile force along an F-actin cable, making a physical barrier separating dorsal and ventral wing disc cells. Navigating the Xenopus embryo (Dev Dyn 2006;235:3059–3062). A frustration shared among Xenopus biologists is their inability to visualize individual cells in the depths of the murky, yolk-filled embryo. Finally, Jacobs' group has found a way to navigate through uncharted territory. They report that two dimensional microscopic magnetic resonance imaging (mMRI) successfully images the orientation, timing, and lineage of cell divisions. Ironically, the often-despised yolk benefits this technique. Its uneven distribution in a cell provides imaging contrast, enabling identification of animal and vegetal poles, blastocele, cell nuclei, and membranes without contrast enhancing labeling. This tool will make the already proven Xenopus model system even more powerful.

Formation of the dorsal marginal zone in Xenopus laevis analyzed by time-lapse microscopic magnetic resonance imaging

The dorsal marginal zone (DMZ) of the amphibian embryo is a key embryonic region involved in body axis organization and neural induction. Using time-lapse microscopic magnetic resonance imaging (MRI), we follow the pregastrula movements that lead to the formation of the DMZ of the stage 10 Xenopus embryo. 2D and 3D MRI time-lapse series reveal that pregastrular movements change the tissue architecture of the DMZ at earlier stages and in a different fashion than previously appreciated. Beginning at stage 9, epiboly of the animal cap moves tissue into the dorsal but not into the ventral marginal zone, resulting in an asymmetry between the dorsal and the ventral sides. Time-lapse imaging of labeled blastomeres shows that the animal cap tissue moves into the superficial DMZ overlying the deeper mesendoderm of the DMZ. The shearing of superficial tissue over the deeper mesendoderm creates the radial/vertical arrangement of ectoderm outside of mesendoderm within the DMZ, which is independent of involution and prior to the formation of the dorsal blastoporal lip. This tilting of the DMZ is distinct from, but occurs synchronously with, the vegetal rotation of the vegetal cell mass [R., Winklbauer, M., Schürfeld (1999). “Vegetal rotation, a new gastrulation movement involved in the internalization of the mesoderm and endoderm in Xenopus.” Development. 126, 3703–3713.]. We present a revised model of gastrulation movements in Xenopus laevis.

Digital Three-Dimensional Atlas of Quail Development Using High-Resolution MRI

We present an archetypal set of three-dimensional digital atlases of the quail embryo based on microscopic magnetic resonance imaging (μMRI). The atlases are composed of three modules: (1) images of fixed ex ovo quail, ranging in age from embryonic day 5 to 10 (e05 to e10); (2) a coarsely delineated anatomical atlas of the μMRI data; and (3) an organ system-based hierarchical graph linked to the anatomical delineations. The atlas is designed to be accessed using SHIVA, a free Java application. The atlas is extensible and can contain other types of information including anatomical, physiological, and functional descriptors. It can also be linked to online resources and references. This digital atlas provides a framework to place various data types, such as gene expression and cell migration data, within the normal three-dimensional anatomy of the developing quail embryo. This provides a method for the analysis and examination of the spatial relationships among the different types of information within the context of the entire embryo.

Two‐dimensional and three‐dimensional time‐lapse microscopic magnetic resonance imaging of Xenopus gastrulation movements using intrinsic tissue‐specific contrast

The amphibian embryo undergoes radical tissue transformations during blastula and gastrula stages, but live observation of internal morphogenetic events by optical microscopy is not feasible due to the opacity of the early embryo. Here, we report on the use of microscopic magnetic resonance imaging (MRI) to directly follow morphogenetic movements during blastula and gastrula stages of the Xenopus laevis embryo. We compare three different MRI modalities that take advantage of the intrinsic contrast present in embryonic tissues: three-dimensional (3D) fat-imaging, 3D water-imaging, and 2D high-speed high-resolution imaging of early embryonic stages. We show that the features revealed by the intrinsic contrast correlate with the histological structure of the embryo. Using this tissue specific intrinsic contrast, the main embryonic tissues and internal tissue movements as well as archenteron invagination can be differentiated without cell labeling. We present 2D and 3D time-lapse sequences of early Xenopus embryonic development, spanning the stages from early blastula to the end of gastrula, which show the complex internal rearrangements of gastrulation in essentially real-time.

Time‐lapse tracing of mitotic cell divisions in the early Xenopus embryo using microscopic MRI

Mitotic cell division is a highly regulated cellular event in all organisms, but its direct visualization in the vertebrates is limited to animals with transparent embryos. Here, we report on the use of microscopic magnetic resonance imaging (mMRI) to noninvasively observe mitotic cell division of early blastomeres in the optically opaque Xenopus laevis embryo. Due to intrinsic tissue contrast, cell nuclei can be directly visualized without the need for contrast enhancing labeling. By taking two-dimensional in vivo time-lapse image sequences, the karyokinesis of a blastomere is followed. Timing and orientation of the cleavages can be traced for five cell divisions to establish a cell lineage tree, including orientation and timing of the mitosis. This work demonstrates for the first time the use of MRI for the visualization of cell divisions and expands the experimental scope of the Xenopus embryo.

Using Magnetic Resonance Microscopy to Assess the Osteogenesis in Porous Hydrogels

AbstractHydrogels are multi-functional and can be used as scaffolds in bone tissue engineering. The pore architecture of the scaffolds is a significant factor in bone cell function. In this work, polyethylene glycol fumarate (OPF) hydrogel is used in the fabrication of porous scaffolds, and the effect of hydrogel porosity on bone formation is evaluated using an in vitro bone marrow stromal cell model. The porous hydrogels consist of copious amounts of water (90% or more by volume), and their structure is very similar to soft tissues. The characterization of porosity and interconnectivity in a typical OPF hydrogel scaffold and the bone formation inside the scaffold are assessed using magnetic resonance microscopy (MRM) techniques; and are shown to be very useful in micro structural studies of such water rich materials. A Bruker Avance 7 Tesla (proton 300 MHz) spectrometer equipped with a Micro Imaging 2.5 accessory is used in these studies. By optimizing the acquisition parameters such as relaxation recovery time (TR), echo time (TE), pulse flip angle (TA), and using solvent relaxation-enhancing media the pores and bone formations are clearly observed at an in plane resolution of 29 μm/pixel in various scaffold materials. Analysis of our MRM images data shows that the pores are highly interconnected and the porosity computed from the images correlate quite well with the experimental porosity parameters such as porogen size and percentage. In addition, the quantitative alkaline phosphatase activity, alizarin red staining as marker of osteoblastic differentiation and the mineralization capacity of marrow stromal cells are in good agreement with the bone formation seen in the MRM images.

Modifications of orientational dependence of microscopic magnetic resonance imaging T2 anisotropy in compressed articular cartilage

To investigate the compression-induced changes in the orientational characteristics in T(2) anisotropy of articular cartilage using microscopic magnetic resonance imaging (microMRI). Six beagle specimens were subjected to various levels of strain (0% to 27%) and were imaged at a minimum of two orientations (0 degrees and 55 degrees ). Two specimens at 14% and 27% strain were imaged at every 5 degrees increment over the first quadrant of the angular space. Quantitative two-dimensional T(2) images and three-dimensional T(2) anisotropy maps of cartilage were constructed at a 19.8-microm in-depth resolution. The load-induced laminar appearance of cartilage at the magic angle became more distinct as the strain level increased. T(2) anisotropy maps of cartilage at 14% and 27% strain exhibited load-induced modifications in the collagen fibril ultrastructure, with a new peak toward the cartilage-bone interface and alterations to orientational dependence of T(2) anisotropy. Distinct alternations in the orientational dependence of microMRI T(2) anisotropy reflect the organizational modification of the collagen matrix due to external loading. This approach could become useful in detecting changes in cartilage's macromolecular structure due to injury or diseases.

A three-dimensional digital atlas database of the adult C57BL/6J mouse brain by magnetic resonance microscopy

A comprehensive three-dimensional digital atlas database of the C57BL/6J mouse brain was developed based on magnetic resonance microscopy images acquired on a 17.6-T superconducting magnet. By using both manual tracing and an atlas-based semi-automatic segmentation approach, T2⁎-weighted magnetic resonance microscopy images of 10 adult male formalin-fixed, excised C57BL/6J mouse brains were segmented into 20 anatomical structures. These structures included the neocortex, hippocampus, amygdala, olfactory bulbs, basal forebrain and septum, caudate-putamen, globus pallidus, thalamus, hypothalamus, central gray, superior colliculi, inferior colliculi, the rest of midbrain, cerebellum, brainstem, corpus callosum/external capsule, internal capsule, anterior commissure, fimbria, and ventricles. The segmentation data were formatted and stored into a database containing three different atlas types: 10 single-specimen brain atlases, an average brain atlas and a probabilistic atlas. Additionally, quantitative group information, such as variations in structural volume, surface area, magnetic resonance microscopy image intensity and local geometry, were computed and stored as an integral part of the database. The database augments ongoing efforts with other high priority strains as defined by the Mouse Phenome Database focused on providing a quantitative framework for accurate mapping of functional, genetic and protein expression patterns acquired by a myriad of technologies and imaging modalities.

The structural adaptations in compressed articular cartilage by microscopic MRI (μMRI) T2 anisotropy

To detect structural adaptations of collagen fiber matrix in compressed articular cartilage in individual sub-tissue zones by microscopic magnetic resonance imaging (microMRI) at 23 microm in-depth resolution. Each of the six beagle humeral cartilage specimens was placed in a specially built nonmetallic compression device inside an in-situ rotation device, and was imaged four times at 7T: without and during static compression, and at two orientations: 0 degrees and 55 degrees . Proton intensity images and quantitative T(2) maps were constructed and analyzed. Upon compression at 55 degrees (the magic angle), rather than appearing homogenous, T(2)-weighted intensity images of cartilage showed a distinct laminar appearance and the T(2) profiles exhibited two distinctive peaks. At both 0 degrees and 55 degrees orientations, lower values of T(2) were observed in compressed tissue. A significant correlation was established between changes in tissue T(2) at 55 degrees and a thickness reduction due to compression. At a mean of 20% strain value, modifications in cartilage structure were studied at each histological zone. We found that the percentage of superficial zone was significantly doubled, the percentage of radial zone was significantly decreased by 10%, and no significant change in the transitional zone. External loading can induce a new kind of laminar appearance at the magic angle. microMRI T(2) anisotropy can be used to analyze the zone-specific alterations in collagen fibril organization in articular cartilage in response to mechanical compression.

Is ‘virtual histology’ the next step after the ‘virtual autopsy’? Magnetic resonance microscopy in forensic medicine

The study aimed to validate magnetic resonance microscopy (MRM) studies of forensic tissue specimens (skin samples with electric injury patterns) against the results from routine histology. Computed tomography and magnetic resonance imaging are fast becoming important tools in clinical and forensic pathology. This study is the first forensic application of MRM to the analysis of electric injury patterns in human skin. Three-dimensional high-resolution MRM images of fixed skin specimens provided a complete 3D view of the damaged tissues at the site of an electric injury as well as in neighboring tissues, consistent with histologic findings. The image intensity of the dermal layer in T2-weighted MRM images was reduced in the central zone due to carbonization or coagulation necrosis and increased in the intermediate zone because of dermal edema. A subjacent blood vessel with an intravascular occlusion supports the hypothesis that current traveled through the vascular system before arcing to ground. High-resolution imaging offers a noninvasive alternative to conventional histology in forensic wound analysis and can be used to perform 3D virtual histology.

Contribution of Magnetic Resonance Microscopy in the 12-Week Neurotoxicity Evaluation of Carbonyl Sulfide in Fischer 344 Rats

In this carbonyl sulfide (COS) study, magnetic resonance microscopy (MRM) and detailed light microscopic evaluation effectively functioned in parallel to assure that the distribution and degree of pathology in the brain was accurately represented. MRM is a powerful imaging modality that allows for excellent identification of neuroanatomical structures coupled with the ability to acquire 200 or more cross-sectional images of the brain, and the ability to display them in multiple planes. F344 rats were exposed to 200-600 ppm COS for up to 12 weeks. Prior to MRM, rats were anesthetized and cardiac perfused with McDowell Trump's fixative containing a gadolinium MR contrast medium. Fixed specimens were scanned at the Duke Center for In Vivo Microscopy on a 9.4 Tesla magnetic resonance system adapted explicitly for microscopic imaging. An advantage of MRM in this study was the ability to identify lesions in rats that appeared clinically normal prior to sacrifice and the opportunity to identify lesions in areas of the brain which would not be included in conventional studies. Other advantages include the ability to examine the brain in multiple planes (transverse, dorsal, sagittal) and obtain and save the MRM images in a digital format that allows for postexperimental data processing and manipulation. MRM images were correlated with neuroanatomical and neuropathological findings. All suspected MRM images were compared to corresponding H&E slides. An important aspect of this study was that MRM was critical in defining our strategy for sectioning the brain, and for designing mechanistic studies (cytochrome oxidase evaluations) and functional assessments (electrophysiology studies) on specifically targeted anatomical sites following COS exposure.

Magnetic resonance microscopy versus light microscopy in human embryology teaching.

A study was carried out on the application of magnetic resonance microscopy (MRM) in teaching prenatal human development. Human embryos measuring 8 mm, 15 mm, 18.5 mm, and 22 mm were fixed in a 4% paraformaldehyde solution and sections obtained with magnetic resonance imaging (MRI) were compared to those prepared for light microscopy (LM), using the same embryos. The MRM and LM slices were of a similar quality. In the MRM sections, embryonic organs and systems were clearly visible, particularly the peripheral and central nervous systems, and the cardiovascular and digestive systems. The digitalization and clarity of the MRM images make them an ideal teaching aid that is suitable for students during the first years of a health-science degree, particularly medicine. As well as providing students with their first experience of MRM, these images allow students to access, at any time, all embryos used, to assess changes in the positions of different organs throughout their stages of development, and to acquire spatial vision, an absolute requirement in the study of human anatomy. We recommend that this technique be incorporated into the wealth of standard embryonic teaching methods already in use.

Bone formation in polymeric scaffolds evaluated by proton magnetic resonance microscopy and X-ray microtomography.

Magnetic resonance microscopy (MRM) and X-ray microtomography (XMT) were used to investigate de novo bone formation in porous poly(ethyl methacrylate) (PEMA) scaffolds, prepared by a novel co-extrusion process. PEMA scaffolds were seeded with primary chick calvarial osteoblasts and cultured under static conditions for up to 8 weeks. Bone formation within porous PEMA scaffolds was confirmed by the application of histologic stains to intact PEMA disks. Disks were treated with Alizarin red to visualize calcium deposits and with Sirius red to visualize regions of collagen deposition. DNA analysis confirmed that cells reached confluence on the scaffolds after 7 weeks in static culture. The formation of bone in PEMA scaffolds was investigated with water proton MRM. Quantitative MRM maps of the magnetization transfer ratio (MTR) yielded maps of protein deposition, and magnetic resonance (MR) relaxation times (T1 and T2) yielded maps of mineral deposition. The location of newly formed bone and local mineral concentrations were confirmed by XMT. By comparing MRM and XMT data from selected regions-of-interest in one sample, the inverse relationship between the MR relaxation times and mineral concentration was validated, and calibration curves for estimating the mineral content of cell-seeded PEMA scaffolds from quantitative MRM images were developed.

Prenatal detection of embryo resorption in osteopontin-deficient mice using serial noninvasive magnetic resonance microscopy.

Appropriate temporal and spatial expression of osteopontin (OPN) in the female genital tract may be critical for successful embryo implantation and maintenance of gestation. Traditionally, experimental assessments of reproductive success have been limited to ex vivo dissection at a single time point to determine embryo number and size and are inadequate for ongoing study of the effect(s) of genetic manipulation on any individual gestation. To investigate the role of OPN in the maintenance of gestation, we developed a noninvasive, in vivo method of pregnancy surveillance suitable for murine application using magnetic resonance microscopy (MRM). Gravid wild-type mice (n =7) and mice with targeted disruption of one or both OPN alleles (OPN(-/-), n = 9; OPN(+/-), n = 3) underwent MRM on postcoital days 10.5, 15.5, and 19.5. Prenatal MRM images were used to determine embryo numbers and sizes. There were no significant differences in embryo numbers determined independently by two blinded observers (mean difference between observers = 0.04 embryos; p = 0.87). There was a significant effect of genotype on embryo size, with OPN(-/-) embryos significantly smaller at all gestational ages. However, targeted disruption of one or more OPN alleles had no effect on embryo number at any gestational age. Thus, MRM may be a powerful noninvasive method for in vivo prenatal developmental study of genetically engineered mice.

Neuroimaging: New Approaches for Neurotoxicology

Over the last 20 years, the impact of imaging on the clinical sciences is unquestionable. It has revolutionized the diagnosis and treatment of disease. Interestingly, the use of imaging in preclinical neurotoxicology has been relatively negligible. This has been in part due to the lack of knowledge or understanding of the capabilities of these powerful technologies. However, some of the more immediately applicable imaging approaches could impact the present approach to neurotoxicology. In addition, the recent advent of the development of imagers specifically for application to small animals will provide the opportunity of obtaining information for neurotoxicological risk assessment in a more timely and relevant manner. The ability to visualize changes in structure and function due to neurotoxic insult in a noninvasive manner is a promising direction. Changes in anatomy of soft and hard tissue, metabolism, function and gene expression can now be done in both a preclinical and a clinical setting using such technologies as magnetic resonance imaging (MRI), magnetic resonance imaging microscopy (MRM), and positron emission tomography (PET). This type of information is not readily accessible using conventional preclinical neurotoxicological procedures and usually requires total destruction of the intrinsic structure of the sample of interest. Imaging provides an opportunity to produce much of these data in a nondestructive manner and presents the data in a three-dimensional format. This permits longitudinal studies of the same subject subsequently reducing the number of animals required for studies while providing more information. In addition, as these technologies have been primarily developed for clinical purposes, they provide an outstanding opportunity for cross-species and animal-to-human extrapolation and testing.

Improved preparation of chick embryonic samples for magnetic resonance microscopy.

Previous work demonstrated the power of three-dimensional (3D) magnetic resonance microscopy (MRM) to follow complicated morphologic development in the embryonic cardiovascular system. In this study we describe a new dual-contrast method for specimen preparation that combines perfusion fixation and immersion in fixative with macro- and small molecular gadolinium agents to provide enhanced definition of both the heart wall and chamber. MRM was performed at 9.4 T with image resolutions of 25, 31, and 50 microm isotropic voxels for three stages of chick embryos (day 4, day 5.5, and day 9), and compared to histological sections of the same embryos. The results show considerable improvement of image quality over previous efforts, with better signal-to-noise ratio (SNR) and contrast between the cardiac chamber and myocardial wall. Excellent correlation was shown between the MRM images and histological sections. Thus, 3D high-resolution MRM in combination with the dual-contrast technique is useful for acquiring quantitative 3D morphologic data regarding heart development.

Imaging the physical and morphological properties of a multi-zone young articular cartilage at microscopic resolution.

To characterize a number of physical and morphologic properties of young articular cartilage. These properties include the anisotropy of T(2) relaxation, optical retardation, orientation of the collagen fibrils, total thickness of the tissue, number of histologic sub-zones in the tissue, width of individual sub-zones, and correlation between the depths of the local T(2) maxima and the local retardation minima. Microscopic magnetic resonance imaging (mciro MRI) and polarized light microscopy (PLM) were used to examine three side-by-side specimens from a humeral head of a three-month-old beagle that exhibited a unique topographic heterogeneity from three-zones centrally to multi-zone peripherally. The centrally located specimen showed that the collagen fibrils across the tissue depth have a pattern of the classic three histologic sub-zones (tangential, transitional, and radial). A much more complicated multi-zone structure was found in the specimen located peripherally, with a second transitional zone and a second tangential zone located at the deep part of the tissue. We also showed that the orientation of the collagen fibrils that form the cocoon-shaped territorial matrix surrounding the clusters of chondrocytes can be imaged by our PLM technique. The results from the young animal in this report, together with our observations from older animals, demonstrate that MRI and PLM can be used to study the epiphyseal expansion of cartilage in young animals during its growth and subsequent loss in older animals. An illustrative model for the structure of collagen fibrils in a humeral head is suggested as an extension to the classic three-zone model for young articular cartilage.

Characteristics of topographical heterogeneity of articular cartilage over the joint surface of a humeral head

Objective To investigate the topographical variations among the morphological and physical properties of articular cartilage over a canine humeral head.Design Nine side-by-side specimens from a canine humeral head were used in the combined polarized light microscopy (PLM) and microscopic magnetic resonance imaging (μMRI). Sixteen images were acquired from each specimen in μMRI. Subsequently, ten to fourteen histological slices were prepared at the location of the μMRI slice from each specimen. Four optical images were acquired from each histological slice. Using these images, the images of the T2 relaxation, the optical retardation, and the collagen-fiber orientation were constructed in two dimensions.Results Along the medial/lateral direction of the humeral head, we have observed a number of topographical variations. These include the total thickness of the cartilage tissue, the thickness of the individual histological zones, the depth of the minimum optical retardation in the tissue, and the depth of the maximum T2 relaxation in the tissue. We found that the depth of the minimum retardation from PLM occurs at the geometric middle point of the transitional zone in histology, and that the depth of the maximum T2 relaxation from μMRI is closely correlated with the minimum retardation depth in PLM. In addition, although the thickness of the total tissue increases approximately by the same amount on both the lateral and medial sides of the joint, a slight asymmetry exists between the measurements from the medial and lateral halves of the humerus.Conclusions Cartilage from different sites over a single joint could have different properties, possibly the consequence of the variation in load-bearing status. Because of these topographical variations, one must keep in mind that a different sampling site within a relatively small area of the same joint may significantly influence the results of the study. Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.

Three-Dimensional Digital Mouse Atlas Using High-Resolution MRI

We present an archetypal digital atlas of the mouse embryo based on microscopic magnetic resonance imaging. The atlas is composed of three modules: (1) images of fixed embryos 6 to 15.5 days postconception (dpc) [Theiler Stages (TS) 8 to 24]; (2) an annotated atlas of the anterior portion of a 13.5 dpc (TS 22) mouse with anatomical structures delineated and linked to explanatory files; and (3) three-dimensional renderings of the entire 13.5 dpc embryo and specific organ systems. The explanatory files include brief descriptions of the structure at each volume element in the image and links to 3D reconstructions, allowing visualization of the shape of the isolated structures. These files can also contain or be linked to other types of information and data including detailed anatomical and physiological information about structures with pointers to online references, relationships between structures, temporal characteristics (cell lineage patterns, size, and shape changes), and gene expression patterns (both spatial and temporal). As an example, we have “painted” in the expression pattern of Dlx5/Dlx6 genes. This digital atlas provides a means to put specific data within the context of normal specimen anatomy, to analyze the information in 3D, and to examine relationships between different types of information.