Digital Volume Data Research Articles

Arterio-venous Anastomoses of the Sucquet-Hoyer Type: Complexity and Distribution in the Human Dermis.

Our study aims at providing detailed information on numbers, form, and spatial distribution of arterio-venous anastomoses of the Sucquet-Hoyer type in the dermis of the nail bed, nail fold corner, thumb pad, arm, nose, glabella, lip, and ear. It further aims at providing a system, which relies on objective morphologic criteria for classifying Sucquet-Hoyer canals (SHCs). Using high-resolution episcopic microscopy (HREM), digital volume data of eight samples of each skin region were produced. Virtual three-dimensional (3D) models of the dermally located SHCs were created, and their 3D tortuosity (τ) values were determined. Dermal SHCs were identified in all 24 finger samples and in 1 lip sample. Beneath a field of 2 × 2 mm2, an average of four were located in the nail bed, three in the dermis of the thumb pad, and one in the dermis of the nail fold corner. Only a single dermal SHC was found in one lip sample. No SHCs were observed in the dermis of the other samples. The τ values of the SHCs ranged from 1.11 to 10. Building on these values, a classification system was designed, which distinguishes four SHC classes. The dermal distribution of the SHCs of different classes was similar in all specimens.

Three-dimensional structural and metric characterisation of cardioids.

Exact three-dimensional (3D) structural information of developing organoids is key for optimising organoid generation and for studying experimental outcomes in organoid models. We set up a 3D imaging technique and studied complexly arranged native and experimentally challenged cardioids of two stages of remodelling. The imaging technique we employed is S-HREM (Scanning High Resolution Episcopic Microscopy), a variant of HREM, which captures multiple images of subsequently exposed surfaces of resin blocks and automatically combines them to large sized digital volume data of voxels sizes below 1μm3. We provide precise volumetric information of the examined specimens and their single components and comparisons between stages in terms of volume and micro- and macroanatomic structure. We describe the 3D arrangement and lining of different types of cavities and their changes between day 10 and day 14 and map the various cell types to their precise spatial and structural environment. Exemplarily, we conducted semiautomatic counts of nuclei. In cryo-injured cardioids, we examined the extension and composition of the injured areas. Our results demonstrate the high quality and the great potential of digital volume data produced with S-HREM. It also provides sound metric and structural information, which assists production of native and experimentally challenged left ventricle cardioids and interpretation of their structural remodelling.

Detailed characterizations of cranial nerve anatomy in E14.5 mouse embryos/fetuses and their use as reference for diagnosing subtle, but potentially lethal malformations in mutants.

Careful phenotype analysis of genetically altered mouse embryos/fetuses is vital for deciphering the function of pre- and perinatally lethal genes. Usually this involves comparing the anatomy of mutants with that of wild types of identical developmental stages. Detailed three dimensional information on regular cranial nerve (CN) anatomy of prenatal mice is very scarce. We therefore set out to provide such information to be used as reference data and selected mutants to demonstrate its potential for diagnosing CN abnormalities. Digital volume data of 152 wild type mice, harvested on embryonic day (E)14.5 and of 18 mutants of the Col4a2, Arid1b, Rpgrip1l and Cc2d2a null lines were examined. The volume data had been created with High Resolution Episcopic Microscopy (HREM) as part of the deciphering the mechanisms of developmental disorders (DMDD) program. Employing volume and surface models, oblique slicing and digital measuring tools, we provide highly detailed anatomic descriptions of the CNs and measurements of the diameter of selected segments. Specifics of the developmental stages of E14.5 mice and anatomic norm variations were acknowledged. Using the provided data as reference enabled us to objectively diagnose CN abnormalities, such as abnormal formation of CN3 (Col4a2), neuroma of the motor portion of CN5 (Arid1b), thinning of CN7 (Rpgrip1l) and abnormal topology of CN12 (Cc2d2a). Although, in a first glimpse perceived as unspectacular, defects of the motor CN5 or CN7, like enlargement or thinning can cause death of newborns, by hindering feeding. Furthermore, abnormal topology of CN12 was recently identified as a highly reliable marker for low penetrating, but potentially lethal defects of the central nervous system.

Artefacts in Volume Data Generated with High Resolution Episcopic Microscopy (HREM).

High resolution episcopic microscopy (HREM) produces digital volume data by physically sectioning histologically processed specimens, while capturing images of the subsequently exposed block faces. Our study aims to systematically define the spectrum of typical artefacts inherent to HREM data and to research their effect on the interpretation of the phenotype of wildtype and mutant mouse embryos. A total of 607 (198 wildtypes, 409 mutants) HREM data sets of mouse embryos harvested at embryonic day (E) 14.5 were systematically and comprehensively examined. The specimens had been processed according to essentially identical protocols. Each data set comprised 2000 to 4000 single digital images. Voxel dimensions were 3 × 3 × 3 µm3. Using 3D volume models and virtual resections, we identified a number of characteristic artefacts and grouped them according to their most likely causality. Furthermore, we highlight those that affect the interpretation of embryo data and provide examples for artefacts mimicking tissue defects and structural pathologies. Our results aid in optimizing specimen preparation and data generation, are vital for the correct interpretation of HREM data and allow distinguishing tissue defects and pathologies from harmless artificial alterations. In particular, they enable correct diagnosis of pathologies in mouse embryos serving as models for deciphering the mechanisms of developmental disorders.

The venous system of E14.5 mouse embryos-reference data and examples for diagnosing malformations in embryos with gene deletions.

Approximately one‐third of randomly produced knockout mouse lines produce homozygous offspring, which fail to survive the perinatal period. The majority of these die around or after embryonic day (E)14.5, presumably from cardiovascular insufficiency. For diagnosing structural abnormalities underlying death and diseases and for researching gene function, the phenotype of these individuals has to be analysed. This makes the creation of reference data, which define normal anatomy and normal variations the highest priority. While such data do exist for the heart and arteries, they are still missing for the venous system. Here we provide high‐quality descriptive and metric information on the normal anatomy of the venous system of E14.5 embryos. Using high‐resolution digital volume data and 3D models from 206 genetically normal embryos, bred on the C57BL/6N background, we present precise descriptive and metric information of the venous system as it presents itself in each of the six developmental stages of E14.5. The resulting data shed new light on the maturation and remodelling of the venous system at transition of embryo to foetal life and provide a reference that can be used for detecting venous abnormalities in mutants. To explore this capacity, we analysed the venous phenotype of embryos from 7 knockout lines (Atp11a, Morc2a, 1700067K01Rik, B9d2, Oaz1, Celf4 and Coro1c). Careful comparisons enabled the diagnosis of not only simple malformations, such as dual inferior vena cava, but also complex and subtle abnormalities, which would have escaped diagnosis in the absence of detailed, stage‐specific referenced data.

High-Resolution Episcopic Microscopy (HREM): Looking Back on 13 Years of Successful Generation of Digital Volume Data of Organic Material for 3D Visualisation and 3D Display

High-resolution episcopic microscopy (HREM) is an imaging technique that permits the simple and rapid generation of three-dimensional (3D) digital volume data of histologically embedded and physically sectioned specimens. The data can be immediately used for high-detail 3D analysis of a broad variety of organic materials with all modern methods of 3D visualisation and display. Since its first description in 2006, HREM has been adopted as a method for exploring organic specimens in many fields of science, and it has recruited a slowly but steadily growing user community. This review aims to briefly introduce the basic principles of HREM data generation and to provide an overview of scientific publications that have been published in the last 13 years involving HREM imaging. The studies to which we refer describe technical details and specimen-specific protocols, and provide examples of the successful use of HREM in biological, biomedical and medical research. Finally, the limitations, potentials and anticipated further improvements are briefly outlined.

The dermal arteries in the cutaneous angiosome of the descending genicular artery.

Studies examining thick skin of the thumb pad have challenged the existence of an arterial plexus in the papillary dermis. Instead of a plexus, discrete arterial units, interconnected by arterio-arterial anastomoses, were identified. We hypothesise that the dermal arteries of thin skin are arranged likewise and that there are fewer arterio-arterial anastomoses in the centre of an angiosome than in zones where neighbouring angiosomes overlap. To test these hypotheses, we examined the dermal arteries in the centre of the cutaneous angiosome of the descending genicular artery (DGA) and its zone of overlap with neighbouring angiosomes. Using traditional perfusion techniques, the cutaneous angiosomes of the DGA and the popliteal artery were identified in 11 fresh frozen human lower limbs. Biopsies were harvested from the centre of the cutaneous DGA angiosome and from the zone where neighbouring vascular territories overlapped. Employing high-resolution episcopic microscopy (HREM), digital volume data were generated and the dermal arteries were three-dimensionally reconstructed and examined. In all examined skin areas, the dermal arteries showed tree-like ramifications. The branches of the dermal arteries were connected on average by 1.73±1.01 arterio-arterial anastomoses in the centre of the DGA angiosome and by 3.27±1.27 in the zone where angiosomes overlapped. We demonstrate that discrete but overlapping dermal arterial units with a mean dimension of 1.62±1.34 and 1.80±1.56mm2 , respectively, supply oxygen and nutrients to the superficial dermis and epidermis of the thin skin of the medial femur. This forms the basis for diagnosing and researching skin pathologies.

Morphology, topology and dimensions of the heart and arteries of genetically normal and mutant mouse embryos at stages S21-S23.

Accurate identification of abnormalities in the mouse embryo depends not only on comparisons with appropriate, developmental stage‐matched controls, but also on an appreciation of the range of anatomical variation that can be expected during normal development. Here we present a morphological, topological and metric analysis of the heart and arteries of mouse embryos harvested on embryonic day (E)14.5, based on digital volume data of whole embryos analysed by high‐resolution episcopic microscopy (HREM). By comparing data from 206 genetically normal embryos, we have analysed the range and frequency of normal anatomical variations in the heart and major arteries across Theiler stages S21–S23. Using this, we have identified abnormalities in these structures among 298 embryos from mutant mouse lines carrying embryonic lethal gene mutations produced for the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme. We present examples of both commonly occurring abnormal phenotypes and novel pathologies that most likely alter haemodynamics in these genetically altered mouse embryos. Our findings offer a reference baseline for identifying accurately abnormalities of the heart and arteries in embryos that have largely completed organogenesis.

High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging organic materials with volumes up to 5 x 5 x 7 mm3 in typical numeric resolutions between 1 x 1 x 1 and 5 x 5 x 5 µm3. Specimens are embedded in methacrylate resin and sectioned on a microtome. After each section an image of the block surface is captured with a digital video camera that sits on the phototube connected to the compound microscope head. The optical axis passes through a green fluorescent protein (GFP) filter cube and is aligned with a position, at which the bock holder arm comes to rest after each section. In this way, a series of inherently aligned digital images, displaying subsequent block surfaces are produced. Loading such an image series in three-dimensional (3D) visualization software facilitates the immediate conversion to digital volume data, which permit virtual sectioning in various orthogonal and oblique planes and the creation of volume and surface rendered computer models. We present three simple, tissue specific protocols for processing various groups of organic specimens, including mouse, chick, quail, frog and zebra fish embryos, human biopsy material, uncoated paper and skin replacement material.

High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials.

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging organic materials with volumes up to 5 x 5 x 7 mm3 in typical numeric resolutions between 1 x 1 x 1 and 5 x 5 x 5 µm3. Specimens are embedded in methacrylate resin and sectioned on a microtome. After each section an image of the block surface is captured with a digital video camera that sits on the phototube connected to the compound microscope head. The optical axis passes through a green fluorescent protein (GFP) filter cube and is aligned with a position, at which the bock holder arm comes to rest after each section. In this way, a series of inherently aligned digital images, displaying subsequent block surfaces are produced. Loading such an image series in three-dimensional (3D) visualization software facilitates the immediate conversion to digital volume data, which permit virtual sectioning in various orthogonal and oblique planes and the creation of volume and surface rendered computer models. We present three simple, tissue specific protocols for processing various groups of organic specimens, including mouse, chick, quail, frog and zebra fish embryos, human biopsy material, uncoated paper and skin replacement material.

High-resolution episcopic microscopy (HREM): A useful technique for research in wound care

Analysing the three-dimensional (3D) texture of skin substitute materials and evaluating their performance after covering skin defects is essential for improving their design and for optimising surgical procedures and post implantation wound treatment regimes. Here we explore the capacities of the recently developed High-resolution episcopic microscopy (HREM) method for generating digital volume data that permit structural 3D analysis of native and implanted collagen–elastin matrices.We employed HREM to visualise native collagen matrices and collagen matrices seeded with keratinocytes. In a second step, we visualised the appearance and the revascularisation of the matrices after their implantation beneath split skin grafts used for covering skin defects in the porcine model. For this, HREM data were generated from biopsies harvested 5, 10, and 15 days after surgery.In all instances, the high quality and resolution of the HREM data in combination with the relative large field of view proved to be sufficient for visualizing the exact fibre architecture by employing quick volume rendering algorithms. Precise analysis of the 3D distribution of keratinocytes in the matrices populated with keratinocytes and of the detailed topology of the sprouting blood vessels in the implanted matrices was feasible.Our results show that high-resolution episcopic microscopy can be adapted to serve as a tool for evaluating collagen–elastin materials ex- and in vivo.

High-resolution episcopic microscopy (HREM): a tool for visualizing skin biopsies.

We evaluate the usefulness of digital volume data produced with the high-resolution episcopic microscopy (HREM) method for visualizing the three-dimensional (3D) arrangement of components of human skin, and present protocols designed for processing skin biopsies for HREM data generation. A total of 328 biopsies collected from normally appearing skin and from a melanocytic nevus were processed. Cuboidal data volumes with side lengths of ~2×3×6 mm3 and voxel sizes of 1.07×1.07×1.5 µm3 were produced. HREM data fit ideally for visualizing the epidermis at large, and for producing highly detailed volume and surface-rendered 3D representations of the dermal and hypodermal components at a structural level. The architecture of the collagen fiber bundles and the spatial distribution of nevus cells can be easily visualized with volume-rendering algorithms. We conclude that HREM has great potential to serve as a routine tool for researching and diagnosing skin pathologies.

The dermal arteries of the human thumb pad

The arteries of the skin have been postulated to form a profound plexus at the dermal/hypodermal junction and a superficial plexus in the papillary dermis. Our article aims to rebut this concept and to provide an alternative description of the arrangement of the dermal arteries. Employing a novel technique, we produced digital volume data (volume size: 2739×2054×3000μm(3) ; voxel size: 1.07×1.07×2μm(3) ) from biopsies of the skin of the thumb pads of 15 body donors. Utilizing these data, we analysed the arrangement of the dermal arteries with the aid of virtual re-sectioning tools, and, in three specimens, with high-quality three-dimensional (3D) surface models. In all specimens we observed a tree-like ramification of discrete dermal arteries. The terminal branches of the arterial trees gave rise to the ascending segments of the capillary loops of the dermal papillae. None of the specimens showed a superficial arterial plexus. This suggests that the skin of the human thumb pad can be split in discrete 'arterial units'. Each unit represents the zone of the papillary dermis and epidermal/dermal junction, to which blood is supplied exclusively by the branches of a single dermal artery. The concept of dermal arterial units is in contrast to all existing descriptions of the architecture of the dermal arteries. However, whether it can be transferred to the skin of other body parts, remains to be tested. Likewise, the consequences of arterial units for understanding the mechanisms of wound healing and the appearance and genesis of skin diseases remain to be examined.

A Chick Embryo With a yet Unclassified Type of Cephalothoracopagus Malformation and a Hypothesis for Explaining its Genesis

Cephalothoracopagus embryos are conjoined twins, who share parts of their heads, necks and bodies. Our study aims at presenting a detailed morphological analysis of a cephalothoracopagus chick embryo of developmental stage 31. Because none of the existing theories can explain the genesis of the phenotype of this embryo, we also suggest a hypothesis, which explains it. Beside the cephalothoracopagus embryo, we investigated five control embryos. With the aid of the high-resolution episcopic microscopy (HREM) technique, we created digital volume data and three-dimensional (3D) computer models of the organs and arteries of the embryos. We used the 3D models for topological analysis and for measuring the diameters of the great intrathoracic arteries. The malformed embryo showed two body backs, each containing a notochord, spinal cord and dorsal aorta. The body backs continued into separated lower bodies. The embryo had a single, four-chambered heart, single respiratory tract and single upper alimentary tract. The topology of the pharyngeal arch arteries was normal, and the diameters of these arteries were similar to that of the control embryos. We classified the embryo we investigated as a yet unknown malformation and suggest a hypothesis explaining its genesis.

Episcopic Three-Dimensional Imaging of Embryos

Episcopic fluorescence image capturing (EFIC) and high-resolution episcopic microscopy (HREM) are related techniques that are used to generate digital volume data and create three-dimensional (3D) images. Both techniques require specimens that are embedded in an appropriate medium, and images are captured from successive sections before removal from the embedded tissue block. EFIC detects autofluorescence emitted from the embedded tissue, whereas HREM requires the tissue to be stained with a fluorescent dye such as eosin. Different procedures are therefore necessary for embedding tissue for EFIC or HREM imaging. This article describes episcopic imaging and gives the advantages and disadvantages of the EFIC and HREM techniques. It also describes the imaging setup required to collect volume data and generate 3D images.

Embedding Embryos for Episcopic Fluorescence Image Capturing (EFIC)

Episcopic fluorescence image capturing (EFIC) and high-resolution episcopic microscopy (HREM) are related techniques that are used to generate digital volume data and create three-dimensional (3D) images. Both techniques require specimens that are embedded in an appropriate medium, and images are captured from successive sections before removal from the embedded tissue block. EFIC detects autofluorescence emitted from the embedded tissue, whereas HREM requires the tissue to be stained with a fluorescent dye such as eosin. Different procedures are therefore necessary for embedding tissue for EFIC or HREM imaging. This protocol describes the procedure for embedding E11.5 mouse embryos for EFIC imaging. Processing times should be adapted for the size and nature of other specimens.

Generation of Volume Data by Episcopic Three-Dimensional Imaging of Embryos: Figure 1.

Episcopic fluorescence image capturing (EFIC) and high-resolution episcopic microscopy (HREM) are related techniques that are used to generate digital volume data and create three-dimensional (3D) images. Both techniques require specimens that are embedded in an appropriate medium, and images are captured from successive sections before removal from the embedded tissue block. EFIC detects autofluorescence emitted from the embedded tissue, whereas HREM requires the tissue to be stained with a fluorescent dye such as eosin. Volume data are generated as the successive sections are imaged and removed using a microtome. This protocol describes the procedure for digital data processing, visualization, and archiving for both EFIC and HREM.

Embedding Embryos for High-Resolution Episcopic Microscopy (HREM)

Episcopic fluorescence image capturing (EFIC) and high-resolution episcopic microscopy (HREM) are related techniques that are used to generate digital volume data and create three-dimensional (3D) images. Both techniques require specimens that are embedded in an appropriate medium, and images are captured from successive sections before removal from the embedded tissue block. EFIC detects autofluorescence emitted from the embedded tissue, whereas HREM requires the tissue to be stained with a fluorescent dye such as eosin. Different procedures are therefore necessary for embedding tissue for EFIC or HREM imaging. For HREM, the choice of fixative appears to be of little consequence. If gene expression patterns are to be visualized in addition to tissue architecture, whole-mount staining for gene expression patterns using the 4-nitro blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) or LacZ detection system must be performed before embedding of samples. This protocol describes the procedure for embedding E11.5 mouse embryos for HREM imaging. Processing times should be adapted for the size and nature of other specimens.

Some Mice Feature 5th Pharyngeal Arch Arteries and Double-Lumen Aortic Arch Malformations

A 5th pair of pharyngeal arch arteries (PAAs) has never been identified with certainty in mice. Murines in general are considered to not develop a 5th pair. If true, the significance of the mouse as a model for researching the genesis of malformations of the great intrathoracic arteries is limited. We aimed to investigate whether mouse embryos develop a 5th pair of PAAs and to identify malformations known to be caused by defective remodelling of the 5th PAAs. We employed the high-resolution episcopic microscopy method for creating digital volume data and three-dimensional (3D) computer models of the great intrathoracic arteries of 30 mouse embryos from days 12–12.5 post conception and 180 mouse fetuses from days 14.5 and 15.5 post conception. The 3D models of the fetuses were screened for the presence of a double-lumen aortic arch malformation. We identified such a malformation in 1 fetus. The 3D models of the embryos were analysed for the presence of 5th PAAs. Six of the 30 embryos (20%) showed a 5th PAA bilaterally, and an additional 9 (30%) showed a 5th PAA unilaterally. Our results prove that some mice do develop a 5th pair of PAAs. They also show that malformations which occur rarely in humans and result from defective remodelling of the left 5th PAA can be identified in mice as well. Thus, the mouse does represent an excellent model for researching the mechanisms driving PAA remodelling and the genesis of malformations of the great intrathoracic arteries.

High-Resolution Episcopic Microscopy Data-Based Measurements of the Arteries of Mouse Embryos: Evaluation of Significance and Reproducibility under Routine Conditions

Defining the role of genes in the genesis of congenital cardiovascular defects involves comparisons of the diameters of arteries measured in wild-type and genetically engineered mouse embryos. This study aims at evaluating the significance and reproducibility of measurements of the diameters of the great intrathoracic arteries of mouse embryos, as produced under routine conditions, by employing a recently suggested measuring method. Using high-resolution episcopic microscopy, we generated digital volume data of 60 mouse embryos (voxel size 1.07 × 1.07 × 2 µm³) of developmental stage 23 according to Theiler. We randomly split the 60 data sets into two groups of 30 and assigned each group to a diploma student. In addition, an experienced scientist received 12 randomly selected specimens of each group. Independently, the researchers created three-dimensional models of the intrathoracic arteries and identified comparable measurement positions along the ascending aorta, pulmonary trunk and descending aorta. At each position, they defined virtual resections cutting through the volume data perpendicular to the longitudinal axis of the artery. In the virtual resections, the researchers measured the perimeter of the lumen of the artery. The diameter was calculated from the perimeter. Then, we performed statistic comparisons of the diameters measured in micrometres and of the ratio of each measured diameter and the diameter of the ascending aorta. Comparisons of the ratios did not reveal statistically significant differences between the measurements created by the different scientists. We assume that the used measuring protocol is highly robust and produces reproducible and significant results under routine conditions.