Quantitative analysis of microscopic X‐ray computed tomography imaging: Japanese quail embryonic soft tissues with iodine staining

Rapid three‐dimensional imaging of embryos to better understand the complex process of morphogenesis has been challenging. Recently introduced iodine staining protocols (I₂KI and alcoholic iodine stains) combined with microscopic X‐ray computed tomography allows visualization of soft tissues in diverse small organisms and tissue specimens. I₂KI protocols have been developed specifically for small animals, with a limited number of quantitative studies of soft tissue contrasts. To take full advantage of the low X‐ray attenuation of ethanol and retain bound iodine while dehydrating the specimen in ethanol, we developed an ethanol I₂KI protocol. We present comparative microscopic X‐ray computed tomography analyses of ethanol I₂KI and I₂KI staining protocols to assess the performance of this new protocol to visualize soft tissue anatomy in late stage Japanese quail embryos using quantitative measurements of soft tissue contrasts and sample shrinkage. Both protocols had only 5% shrinkage compared with the original harvested specimen, supporting the use of whole mounts to minimize tissue shrinkage effects. Discrimination within and among the selected organs with each staining protocol and microscopic X‐ray computed tomography imaging were comparable to those of a gray scale histological section. Tissue discrimination was assessed using calibrated computed tomography values and a new discrimination index to quantify the degree of computed tomography value overlaps between selected soft tissue regions. Tissue contrasts were dependent on the depth of the tissue within the embryos before the embryos were saturated with each stain solution, and optimal stain saturations for the entire embryo were achieved at 14 and 28 days staining for I₂KI and ethanol I₂KI, respectively. Ethanol I₂KI provided superior soft tissue contrasts by reducing overstaining of fluid‐filled spaces and differentially modulating staining of some tissues, such as bronchial and esophageal walls and spinal cord. Delineating the selected soft tissues using optimal threshold ranges derived from the quantitative analyses of the contrast enhancement in optimally stained embryos is possible. The protocols presented here are expected to be applicable to other organisms with modifications to staining time and contribute toward rapid and more efficient segmentation of soft tissues for three‐dimensional visualization.     

Three‐dimensional imaging of the fibrous microstructure of Achilles tendon entheses in Mus musculus

The whole‐organ, three‐dimensional microstructure of murine Achilles tendon entheses was visualized with micro‐computed tomography (microCT). Contrast‐enhancement was achieved either by staining with phosphotungstic acid (PTA) or by a combination of cell‐maceration, demineralization and critical‐point drying with low tube voltages and propagation‐based phase‐contrast (fibrous structure scan). By PTA‐staining, X‐ray absorption of the enthesial soft tissues became sufficiently high to segment the tendon and measure cross‐sectional areas along its course. With the fibrous structure scans, three‐dimensional visualizations of the collagen fiber networks of complete entheses were obtained. The characteristic tissues of entheses were identified in the volume data. The tendon proper was marked as a segment manually. The fibers within the tendon were marked by thresholding. Tendon and fiber cross‐sectional areas were measured. The measurements were compared between individuals and protocols for contrast‐enhancement, using a spatial reference system within the three‐dimensional enthesis. The usefulness of the method for investigations of the fibrous structure of collagenous tissues is demonstrated.     

High‐resolution MRI of early‐stage mouse embryos

Both the availability of methods to manipulate genes and the completion of the mouse genome sequence have led to the generation of thousands of genetically modified mouse lines that provide a new platform for the study of mammalian development and developmental diseases. Phenotyping of mouse embryos has traditionally been performed on fixed embryos by the use of ex vivo histological, optical and high‐resolution MRI techniques. Although potentially powerful, longitudinal imaging of individual animals is difficult or impossible with conventional optical methods because of the inaccessibility of mouse embryos inside the maternal uterus. To address this problem, we present a method of imaging the mouse embryo from stages as early as embryonic day (E)10.5, close to the onset of organogenesis in most physiological systems. This method uses a self‐gated MRI protocol, combined with image registration, to obtain whole‐embryo high‐resolution (100 µm isotropic) three‐dimensional images. Using this approach, we demonstrate high contrast in the cerebral vasculature, limbs, spine and central nervous system without the use of contrast agents. These results indicate the potential of MRI for the longitudinal imaging of developing mouse embryos in utero and for future applications in analyzing mutant mouse phenotypes. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative three‐dimensional imaging of live avian embryonic morphogenesis via micro‐computed tomography

Many clinically relevant congenital malformations arise during mid to late embryonic stages. This period is challenging to image quantitatively in live embryos, necessitating the use of multiple specimens with increased experimental variability. Here we establish X‐ray and blood‐pool computed tomography (CT) contrast agent toxicity and teratogenesis thresholds for 3D Micro‐CT imaging of live avian embryos. Day 4 chick embryos micro‐injected with Visipaque? (VP) developed for an additional 6 days without defect. X‐ray radiation up to 798 mGy was nontoxic. Peak average contrast of 1,060 HU occurred within 1 hr of imaging at 50 μm resolution. VP‐enhanced contrast persisted past 24 hr with delayed accumulation in the allantois. Regional volumes of VP‐injected embryos were statistically identical to those of fixed embryos perfused with osmium tetroxide. We further quantified longitudinal volumetric morphogenesis of the allantois over 30 hr. These results demonstrate the safety and efficacy of contrast enhanced quantitative micro‐CT imaging for live embryos. Developmental Dynamics 240:1949–1957, 2011. © 2011 Wiley‐Liss, Inc.     

Diffusible iodine‐based contrast‐enhanced computed tomography (diceCT): an emerging tool for rapid,high‐resolution, 3‐D imaging of metazoan soft tissues

Morphologists have historically had to rely on destructive procedures to visualize the three‐dimensional (3‐D) anatomy of animals. More recently, however, non‐destructive techniques have come to the forefront. These include X‐ray computed tomography (CT), which has been used most commonly to examine the mineralized, hard‐tissue anatomy of living and fossil metazoans. One relatively new and potentially transformative aspect of current CT‐based research is the use of chemical agents to render visible, and differentiate between, soft‐tissue structures in X‐ray images. Specifically, iodine has emerged as one of the most widely used of these contrast agents among animal morphologists due to its ease of handling, cost effectiveness, and differential affinities for major types of soft tissues. The rapid adoption of iodine‐based contrast agents has resulted in a proliferation of distinct specimen preparations and scanning parameter choices, as well as an increasing variety of imaging hardware and software preferences. Here we provide a critical review of the recent contributions to iodine‐based, contrast‐enhanced CT research to enable researchers just beginning to employ contrast enhancement to make sense of this complex new landscape of methodologies. We provide a detailed summary of recent case studies, assess factors that govern success at each step of the specimen storage, preparation, and imaging processes, and make recommendations for standardizing both techniques and reporting practices. Finally, we discuss potential cutting‐edge applications of diffusible iodine‐based contrast‐enhanced computed tomography (diceCT) and the issues that must still be overcome to facilitate the broader adoption of diceCT going forward.     

Micro‐Computed Tomography for Visualizing Limb Skeletal Regeneration in Young Xenopus Frogs

For studies of vertebrate limb regeneration it is often desirable to visualize the regenerated skeleton, which is mostly cartilage, and also section the specimen for histological or immunohistochemical visualization of other tissues. However, the normal skeletal staining techniques are incompatible with immunohistochemistry. Here, we describe a contrast‐based micro‐computed tomography (microCT) method for direct and nondestructive observation of regenerated cartilage spikes in Xenopus frog limbs. In addition, we show that contrast based microCT imaging is compatible with immunohistochemistry protocols. This approach provides versatile and high contrast images of the cartilage allowing us to measure the regenerated skeletal structure in detail as well as carrying out the other types of analysis. It opens a wide range of potential microCT applications in research on vertebrate limb regeneration. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

MicroCT for molecular imaging: quantitative visualization of complete three-dimensional distributions of gene products in embryonic limbs

We present a broadly applicable procedure for whole-mount imaging of antibody probes in embryonic tissues at microscopic resolutions based on combining a metal-based immunodetection scheme with x-ray microtomography (microCT). The method is generally accessible, relying on standard enzyme-conjugated secondary antibodies and other readily available reagents, and is demonstrated here with microCT visualizations of acetylated α-tubulin in the chick nervous system and of type II collagen in developing limbs. The tomographic images offer complete three-dimensional representations of molecular patterns obtained with immunostaining methods at the level of organ development, with added possibilities to quantify both spatial distributions and varying densities of gene products in situ. This imaging modality bridges a crucial gap in three-dimensional molecular imaging by combining the histological resolutions of confocal microscopy with a greater specimen size range than optical projection tomography, and thus enables a powerful new approach to long-standing issues of skeletogenic pattern formation in vertebrate limbs.     

Technique: Imaging Earliest Tooth Development in 3D Using a Silver‐Based Tissue Contrast Agent

Looking in microscopic detail at the 3D organization of initiating teeth within the embryonic jaw has long‐proved technologically challenging because of the radio‐translucency of these tiny un‐mineralized oral tissues. Yet 3D image data showing changes in the physical relationships among developing tooth and jaw tissues are vital to understand the coordinated morphogenesis of vertebrate teeth and jaws as an animal grows and as species evolve. Here, we present a new synchrotron‐based scanning solution to image odontogenesis in 3D and in histological detail using a silver‐based contrast agent. We stained fixed, intact wild‐type mice aged embryonic (E) day 10 to birth with 1% Protargol‐S at 37°C for 12–32 hr. Specimens were scanned at 4–10 µm pixel size at 28 keV, just above the silver K‐edge, using micro‐computed tomography (µCT) at the Canadian Light Source synchrotron. Synchrotron µCT scans of silver‐stained embryos showed even the earliest visible stages of tooth initiation, as well as many other tissue types and structures, in histological detail. Silver stain penetration was optimal for imaging structures in intact embryos E15 and younger. This silver stain method offers a powerful yet straightforward approach to visualize at high‐resolution and in 3D the earliest stages of odontogenesis in situ, and demonstrates the important of studying the tooth organ in all three planes of view. Anat Rec, 297:222–233, 2014. © 2013 Wiley Periodicals, Inc.     

In vivo photoacoustic imaging of mouse embryos

The ability to noninvasively image embryonic vascular anatomy in mouse models is an important requirement for characterizing the development of the normal cardiovascular system and malformations in the heart and vascular supply. Photoacoustic imaging, which can provide high resolution non invasive images of the vasculature based upon optical absorption by endogenous hemoglobin, is well suited to this application. In this study, photoacoustic images of mouse embryos were obtained ex vivo and in vivo. The images show intricate details of the embryonic vascular system to depths of up to 10 mm, which allowed whole embryos to be imaged in situ. To achieve this, an all-optical photoacoustic scanner and a novel time reversal image reconstruction algorithm, which provide deep tissue imaging capability while maintaining high spatial resolution and contrast were employed. This technology may find application as an imaging tool for preclinical embryo studies in developmental biology as well as more generally in preclinical and clinical medicine for studying pathologies characterized by changes in the vasculature.     

Optimization of Volumetric Computed Tomography for Skeletal Analysis of Model Genetic Organisms

Forward and reverse genetics now allow researchers to understand embryonic and postnatal gene function in a broad range of species. Although some genetic mutations cause obvious morphological change, other mutations can be more subtle and, without adequate observation and quantification, might be overlooked. For the increasing number of genetic model organisms examined by the growing field of phenomics, standardized but sensitive methods for quantitative analysis need to be incorporated into routine practice to effectively acquire and analyze ever‐increasing quantities of phenotypic data. In this study, we present platform‐independent parameters for the use of microscopic x‐ray computed tomography (microCT) for phenotyping species‐specific skeletal morphology of a variety of different genetic model organisms. We show that microCT is suitable for phenotypic characterization for prenatal and postnatal specimens across multiple species. Anat Rec, 291:475–487, 2008. © 2008 Wiley‐Liss, Inc.     

Direct MicroCT imaging of non-mineralized connective tissues at high resolution

The 3D imaging of soft tissues in their native state is challenging, especially when high resolution is required. An X-ray-based microCT is, to date, the best choice for high resolution 3D imaging of soft tissues. However, since X-ray attenuation of soft tissues is very low, contrasting enhancement using different staining materials is needed. The staining procedure, which also usually involves tissue fixation, causes unwanted and to some extent unknown tissue alterations. Here, we demonstrate that a method that enables 3D imaging of soft tissues without fixing and staining using an X-ray-based bench-top microCT can be applied to a variety of different tissues. With the sample mounted in a custom-made loading device inside a humidity chamber, we obtained soft tissue contrast and generated 3D images of fresh, soft tissues with a resolution of 1 micron voxel size. We identified three critical conditions which make it possible to image soft tissues: humidified environment, mechanical stabilization of the sample and phase enhancement. We demonstrate the capability of the technique using different specimens: an intervertebral disc, the non-mineralized growth plate, stingray tessellated radials (calcified cartilage) and the collagenous network of the periodontal ligament. Since the scanned specimen is fresh an interesting advantage of this technique is the ability to scan a specimen under load and track the changes of the different structures. This method offers a unique opportunity for obtaining valuable insights into 3D structure–function relationships of soft tissues.     

In‐laboratory diffraction‐enhanced X‐ray imaging for articular cartilage

The loss of articular cartilage characteristic of osteoarthritis can only be diagnosed by joint space narrowing when conventional radiography is used. This is due to the lack of X‐ray contrast of soft tissues. Whereas conventional radiography harnesses the X‐ray attenuation properties of tissues, Diffraction Enhanced Imaging (DEI), a novel radiographic technique, allows the visualization of soft tissues simultaneous with calcified tissues by virtue of its ability to not only harness X‐ray attenuation but also the X‐ray refraction from tissue boundaries. Previously, DEI was dependent upon synchrotron X‐rays, but more recently, the development of nonsynchrotron DEI units has been explored. These developments serve to elaborate the full potential of radiography. Here, we tested the potential of an in‐laboratory DEI system, called Diffraction‐Enhanced X‐ray Imaging (DEXI), to render images of articular cartilage displaying varying degrees of degradation, ex vivo. DEXI allowed visualization of even early stages of cartilage degeneration such as surface fibrillation. This may be of eventual clinical significance for the diagnosis of early stages of degeneration, or at the very least, to visualize soft tissue degeneration simultaneous with bone changes. Clin. Anat. 23:530–538, 2010. © 2010 Wiley‐Liss, Inc.     

Automated image‐based phenotypic analysis in zebrafish embryos

Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high‐throughput chemical screens, and optical transparency makes them potentially suited for image‐based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to using the zebrafish as a high‐throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi‐well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high‐content reader and analyzed using an artificial intelligence‐based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP)^y1) arrayed in 96‐well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image‐based high‐content screening methodology to measure complex whole organism phenotypes. Developmental Dynamics 238:656–663, 2009. © 2009 Wiley‐Liss, Inc.     

Developmental atlas of the early first trimester human embryo

Rapid advances in medical imaging are facilitating the clinical assessment of first‐trimester human embryos at increasingly earlier stages. To obtain data on early human development, we used magnetic resonance (MR) imaging and episcopic fluorescence capture (EFIC) to acquire digital images of human embryos spanning the time of dynamic tissue remodeling and organogenesis (Carnegie stages 13 to 23). These imaging data sets are readily resectioned digitally in arbitrary planes, suitable for rapid high‐resolution three‐dimensional (3D) observation. Using these imaging datasets, a web‐accessible digital Human Embryo Atlas ( http://apps.devbio.pitt.edu/humanatlas/ ) was created containing serial 2D images of human embryos in three standard histological planes: sagittal, frontal, and transverse. In addition, annotations and 3D reconstructions were generated for visualizing different anatomical structures. Overall, this Human Embryo Atlas is a unique resource that provides morphologic data of human developmental anatomy that can accelerate basic research investigations into developmental mechanisms that underlie human congenital anomalies. Developmental Dynamics 239:1585–1595, 2010. © 2010 Wiley‐Liss, Inc.     

Postnatal Mandibular Cheek Tooth Development in the Miniature Pig Based on Two‐Dimensional and Three‐Dimensional X‐Ray Analyses

The miniature pig is a useful large laboratory animal model. Various tissues and organs of miniature pigs are similar to those of humans in terms of developmental, anatomical, immunological, and physiological characteristics. The oral and maxillofacial region of miniature pigs is often used in preclinical studies of regenerative dentistry. However, there is limited information on the dentition and tooth structure of miniature pigs. The purpose of this study was to examine the time‐course changes of dentition and tooth structure (especially the root) of the miniature pig mandibular cheek teeth through X‐ray analyses using soft X‐ray for two‐dimensional observations and micro‐CT for three‐dimensional observations. The mandibles of male Clawn strain miniature pigs (2 weeks and 3, 5, 7, 9, 11, 14, 17, and 29 months of age) were used. X‐ray analysis of the dentition of miniature pig cheek teeth showed that the eruption pattern of the miniature pig is diphyodont and that the replacement pattern is vertical. Previous definitions of deciduous and permanent teeth often varied and there has been no consensus on the number of teeth (dentition); however, we found that three molars are present in the deciduous dentition and that four premolars and three molars are present in the permanent dentition. Furthermore, we confirmed the number of tooth roots and root canals. We believe that these findings will be highly useful in future studies using miniature pig teeth. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.     

Prolonged in vivo imaging of Xenopus laevis

Background: While live imaging of embryonic development over long periods of time is a well established method for embryos of the frog Xenopus laevis, once development has progressed to the swimming stages, continuous live imaging becomes more challenging because the tadpoles must be immobilized. Current imaging techniques for these advanced stages generally require bringing the tadpoles in and out of anesthesia for short imaging sessions at selected time points, severely limiting the resolution of the data. Results: Here we demonstrate that creating a constant flow of diluted tricaine methanesulfonate (MS‐222) over a tadpole greatly improves their survival under anesthesia. Based on this result, we describe a new method for imaging stage 48 to 65 X. laevis, by circulating the anesthetic using a peristaltic pump. This supports the animal during continuous live imaging sessions for at least 48 hr. The addition of a stable optical window allows for high quality imaging through the anesthetic solution. Conclusions: This automated imaging system provides for the first time a method for continuous observations of developmental and regenerative processes in advanced stages of Xenopus over 2 days. Developmental Dynamics 243:1011–1019, 2014. © 2014 Wiley Periodicals, Inc.     

Preparation of PEGylated Iodine‐Loaded Nanoparticles via Polymer‐Directed Self‐Assembly

The preparation of size‐tunable PEGylated, iodine‐loaded nanoparticles is investigated for biomedical applications. Di‐iodination of polyvinyl phenol and encapsulation of the iodinated polymer via directed self‐assembly with an amphiphilic polyethylene glycol‐based diblock copolymer are reported. Nanoparticles with iodine loadings up to 45 wt% are achieved using a rapid, scalable process. The size of the nanoparticles can be readily tuned between 35 and 130 nm by increasing the ionic strength of the antisolvent used during nanoparticle self‐assembly. The resulting PEGylated iodine‐loaded nanoparticles have potential applications in nanomedicine for 1) quantitative biodistribution analysis via inductively coupled plasma mass spectrometry (ICP‐MS) or 2) X‐ray contrast in biomedical imaging. For quantitative biodistribution studies using ICP‐MS, a limit of detection of 2 µg mL^?1 in mouse serum is achieved. For biomedical imaging, the X‐ray attenuation rates are comparable to currently commercially available iodine‐based contrast agents. Therefore, encapsulation of the iodinated polymer enables formulation of trackable, size tunable nanoparticles as a versatile platform for developing nanomedicines.     

Surface imaging microscopy, an automated method for visualizing whole embryo samples in three dimensions at high resolution.

Modern biology is faced with the challenge of understanding the specification, generation, and maintenance of structures ranging from cells and tissues to organs and organisms. By acquiring images directly from the block face of an embedded sample, surface imaging microscopy (SIM) generates high-resolution volumetric images of biological specimens across all of these scales. Surface imaging microscopy expands our range of imaging tools by generating three-dimensional reconstructions of embryo samples at high resolution and high contrast. SIM image quality is not limited by depth or the optical properties of overlying tissue, and intrinsic or extrinsic alignment markers are not required for volume reconstruction. These volumes are highly isotropic, enabling them to be virtually sectioned in any direction without loss of image quality. Surface imaging microscopy provided a more accurate three-dimensional representation of a chick embryo than confocal microscopy of the same sample. SIM offers excellent imaging of embryos from three major vertebrate systems in developmental biology: mouse, chicken, and frog. Immediate applications of this technology are in visualizing and understanding complex morphogenetic events and in making detailed comparisons between normal and genetically modified embryos.     

A staging system for correct phenotype interpretation of mouse embryos harvested on embryonic day 14 (E14.5)

We present a simple and quick system for accurately scoring the developmental progress of mouse embryos harvested on embryonic day 14 (E14.5). Based solely on the external appearance of the maturing forelimb, we provide a convenient way to distinguish six developmental sub‐stages. Using a variety of objective morphometric data obtained from the commonly used C57BL/6N mouse strain, we show that these stages correlate precisely with the growth of the entire embryo and its organs. Applying the new staging system to phenotype analyses of E14.5 embryos of 58 embryonic lethal null mutant lines from the DMDD research programme ( https://dmdd.org.uk ) and its pilot, we show that homozygous mutant embryos are frequently delayed in development. To demonstrate the importance of our staging system for correct phenotype interpretation, we describe stage‐specific changes of the palate, heart and gut, and provide examples in which correct diagnosis of malformations relies on correct staging.