Studies in anatomy are frequently performed through dissection and histology as investigative methods. These techniques are, however, destructive and struggle to capture the peripheral nerves over larger areas, such as the pelvis, and their trajectories thus remain elusive. This study aimed to explore the capabilities of non-destructive micro-CT in identifying small nerves in the periphery of the pelvic area and map them in human fetal development. Six human fetal pelvic samples were derived from the Dutch Fetal Biobank; samples were contrast enhanced with B-Lugol and scanned through micro-CT imaging. Scans were segmented using Avizo Amira software to quantify nerves entering the rectum by crossing the longitudinal muscle layer and visualize their trajectory in relationship to pelvic organs across development age range and sex. Results were verified with haematoxylin and eosin stained histological slices. The following conclusions were found: (1) Micro-CT is able to resolve nerves with a diameter larger than 60 μm in the periphery of the pelvic area in full fetal pelvises of 4 cm in size. (2) Both above and below the level of the levator ani, small lateral nerves enter the anal canal by penetrating the longitudinal muscle layer. (3) Males appear to have more crossing nerves than females. (4) A developmental shift was observed with more nerve crossings below the levator ani muscle in older fetuses. Micro-CT was successfully applied to scan human fetal pelvic samples to study nerves and provides a new way to analyse topographical nerve development. Microscale nerves cross to the anal canal at all levels, which should be taken into account when operating in this area.

We present the first application of Hierarchical Phase-Contrast Tomography (HiP-CT) to complete human fetuses at BM18, the flagship beamline for microtomography on large samples at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Leveraging the advanced capabilities of BM18, six human fetal specimens from the Dutch Fetal Biobank (Amsterdam UMC, ethics approval 2016_285) were scanned at multiple resolutions. This pilot cohort included two specimens at approximately 21 weeks of gestation (one with a cardiac anomaly), two at 16 weeks (one with a cardiac anomaly), and two at 11 weeks (including conjoined twins).The imaging pipeline comprised rapid post-mortem tissue recovery (2–6 h interval), paraformaldehyde fixation, transfer to 70% ethanol, mounting, degassing in custom vacuum chambers, and transport from Amsterdam UMC to ESRF. Initial whole-body overviews were acquired at 16 or 8 µm isotropic voxel size, followed by targeted scans at 4 and 2 µm, yielding dozens of terabytes of data. Thanks to the high sensitivity brought by the HiP-CT protocols, these multiscale datasets provide unprecedented structural detail and enable exploration of complex anatomical systems including the lymphatic and nervous systems, fascia, and inter-organ relationships. Post-acquisition workflows included advanced segmentation pipelines to support quantitative analysis and AI model training.To our knowledge, this is the first time the human body has been imaged in its entirety at such high resolution. This pilot study marks a critical expansion of the BM18 HiP-CT pipeline into the fetal domain. These data hold wide-ranging implications for anatomy, developmental biology, congenital anomaly research, and forensic applications. Future steps include cross-institutional collaboration for quantitative analysis and integration with downstream histology and spatial transcriptomics.

Background The primary ossification of skeletal structures occurs predominantly in-utero. For this reason, knowledge of skeletal development is based on clinical imaging methods (e.g. ultrasound, or in rare cases MRI), or derived from archaeological or forensic specimens with an unknown fetal age. To accurately estimate age-at-death in forensic cases, further knowledge of skeletal development is essential. Methods Thirty-four healthy human fetuses (9-24 weeks gestational age) previously donated to the Dutch Fetal Biobank after termination of pregnancy have undergone diffusible iodine-based contrast enhancement and micro-CT scanning. All cartilaginous and ossified skeletal elements were first segmented using in-house-developed AI tools, after which the segmentations were manually verified and adjusted in Dragonfly 3D World. Additional micro-CT scans without contrast enhancement of healthy human fetuses were used to further characterize the timing of ossification onset and to quantify the size and morphology of primary ossification centres. Expected Results We will provide quantifications and morphological visualisations of the developing human fetus. Growth curves with prediction intervals will be developed based on size and ossification onsets for all major long bones, providing robust quantitative references for early skeletal development. Also, the size and morphology of primary ossification centres together with the timing of ossification onset will be characterized over gestational ages. Conclusions With this project we will develop a comprehensive source on fetal skeletal growth and development, providing three-dimensional morphological visualisations, ossification onsets, and growth curves of human fetuses at nine to 24 weeks of gestation.

The Human Organ Atlas Hub (HOAHub) is an international consortium dedicated to mapping complete human organs in unprecedented three-dimensional detail. Using Hierarchical Phase-Contrast Tomography (HiP-CT), developed with ESRF at beamlines BM18 and BM05, entire human organs can now be imaged non-destructively across multiple resolution scales—from whole-organ overviews at 20 µm to sub-micrometer virtual 3D histology. This multiscale approach opens new avenues for forensic imaging by enabling structural assessment at resolutions traditionally achievable only through destructive sampling.At Amsterdam UMC we have spent the past two years building a robust organ-processing pipeline that maximizes tissue quality for HiP-CT. Thanks to a dedicated 24/7 rapid-recovery program, organs from body donors can be obtained within a 2–6-hour post-mortem interval, preserving fine structural integrity. Immediate fixation in fresh paraformaldehyde ensures sample stability while still allowing downstream correlation with classical histology and emerging molecular techniques such as spatial transcriptomics—critical for studying tissue responses, disease processes, or injury patterns.In our laboratory, organs are mounted and carefully degassed in a custom vacuum chamber to ensure optimal X-ray transparency before transport to ESRF. Following HiP-CT scanning, datasets undergo advanced computational processing. Students and researchers generate extensive ground-truth segmentations to train AI models capable of automatically identifying key anatomical structures, including vessels and nerves.Since September, Amsterdam UMC hosts a large-format Micro-CT system, allowing us to scan much larger volumes of patient material. The ESRF HiP-CT datasets serve as the gold-standard reference, enhancing interpretation of Micro-CT findings and supporting the development of improved protocols for post-mortem imaging.Together, these innovations form an integrated pipeline with strong potential for forensic applications, including improved injury characterization, anatomically precise documentation, and more accurate reconstructions of pathological or traumatic processes.

Background Traditional postmortem fingerprinting is often unreliable in cases of decomposition or thermal trauma. Although friction ridge patterns are typically recorded from the epidermis, their anatomical origin in the dermis may preserve identifiable structures, such as friction ridges and sweat pores, even when the skin's outer layer is compromised. Minimally destructive postmortem imaging techniques capable of visualizing these subsurface structures could therefore greatly enhance forensic identification. Objective This pilot study explores the feasibility of micro-computed tomography (micro-CT) as a novel, minimally destructive technique for visualizing subsurface fingerprint structures in challenging postmortem forensic contexts. Methods Thirteen human finger samples in various postmortem conditions (fresh, decomposed and burned) were included. Four were scanned using micro-CT. Contrast staining was selectively applied. Micro-CT findings were descriptively contextualized using conventional postmortem fingerprinting techniques. Results Micro-CT imaging successfully visualized friction ridges and sweat gland structures in fresh samples, including at lower scanning resolutions. In decomposed samples, micro-CT imaging revealed little anatomical detail, whereas warm water rehydration restored usable ridge patterns. In burned samples, micro-CT imaging failed to recover ridge detail but visualized sweat gland structures. Conclusion Micro-CT imaging shows potential as a complementary tool for postmortem identification in burned or fresh remains but provides limited additional value in cases of advanced decomposition. As a pilot study, these findings primarily provide anatomical and technical insights. Future studies should explore larger sample sizes, sweat gland tracing and integration with 3D matching software.

Objectives Hierarchical Phase-Contrast Tomography (HiP-CT) enables non-destructive, multi-scale imaging of whole human organs. We describe how HiP-CT is utilized for biomedical research within the Human Organ Atlas Hub through three case studies: mapping the enteric nervous system (ENS) of the human colon, analysing myocardial and AV conduction architecture in Tetralogy of Fallot (TOF), and characterizing ductal organization in breast carcinoma. The challenges we faced with this novel biomedical data are discussed. Methods Whole-organ and region-of-interest scans of three types of human organs were acquired at the European Synchrotron Radiation Facility (ESRF) with isotropic voxel sizes ranging from 20 um to 0.8 um. For the colon, voxel binning and RootPainter were employed to tackle data size to segment the ENS. For the heart, voxel-wise myocyte orientation mapping was calculated in terabyte-scale datasets with a high-performance computational framework (Cardiotensor). Breast carcinoma samples were correlated with histopathology for structure validation. Results HiP-CT revealed the large-scale organization of the ENS in the colon, enabling visualisation of the 3D structures of the ENS across the colon In TOF hearts, analysis uncovered abnormal myocardial structure and heterogeneous conduction system morphology. In breast carcinoma, HiP-CT resolved the full hierarchy of ductal structures and vascular relationships within tumour and peritumoral regions. Conclusions HiP-CT provides unprecedented, hierarchical insight into intact human organ structure, bridging the gap between histology and radiology. Advances in knowledge HiP-CT establishes a new ex vivo radiological modality capable of linking microscale pathology to whole-organ context, advancing translational research in neurogastroenterology, cardiology, and oncology ### Competing Interest Statement The authors have declared no competing interest. Chan Zuckerberg Initiative (United States), 2022‑316777 Wellcome Trust, https://ror.org/029chgv08, 310796/Z/24/Z RAEng Chair in Emerging Technologies, CiET1819/10 CIFAR Catalyst Award

Background and Aims Patients born with anorectal malformations (ARM) might experience constipation and fecal incontinence. During ARM surgery (A/PSARP procedure), the distal part of the bowel (fistula) is usually resected. Micro-CT imaging, capable of imaging samples in ultra-high 3D resolution, can be utilized to learn from this resected material. Through this technique we aim to investigate whether or not structures, such as the internal anal sphincter (IAS), are present within this fistula. Methods Pediatric patients undergoing surgical reconstruction for ARM were eligible for inclusion. Resected fistulas were fixed using 4% paraformaldehyde (PFA) and stained with 3.75% B-Lugol for 48 hours to improve soft tissue contrast. Scans were performed on a Phoenix Nanotom micro-CT with a voxel size between 4-6 μm. Samples were destained for subsequent histopathological examination. Outcomes were presence of structures like the IAS, epithelial transition zone and ganglia. ARM fistulas were compared with a fetal anal canal sample derived from the Dutch Fetal Biobank. Results 11 ARM fistulas were analysed. All samples showed evidence of normal development of the rectal wall. Columnar epithelium and stratified squamous epithelium were observed. Muscle fibres were present, arranged in circular pattern that expanded towards the distal end, becoming the intrinsic sphincter (IAS). Ganglia were present with normal appearance. Conclusion We present micro-CT imaging to research resected material to provide new insights in microscale anatomy. The fistula, currently resected during surgical reconstruction for ARM, contains vital structures like the IAS, normal epithelial transition zone and normal ganglion cells. Although clinical functionality should be studied in the future, our results indicate that the fistula has a normal anal canal morphology and should be spared during ARM reconstruction if possible.

AIM:Forensic examination of the hyoid-larynx complex (HLC) is crucial in diagnosing (inflicted) neck trauma. Clinical Total-Body CT (TBCT) scans of the (non-)calcified HLC lack sensitivity for fracture detection. Micro-computed tomography (micro-CT) is an upcoming imaging modality capable of scanning samples at microscale resolution up to 1 µm. Soft-tissue contrast can be established with iodine staining, known as diffusible iodine-based contrast-enhanced CT (diceCT). We study the additional value of micro- and diceCT in detecting HLC fractures and hemorrhages in forensic cases, compared to standard forensic imaging techniques and findings at autopsy. MATERIALS AND METHODS:Fifteen, during forensic autopsy excised, adult HLCs were scanned with micro-CT. Subsequently, the samples were stained with buffered Lugol's solution (B-Lugol) and scanned with contrast-enhanced micro-CT. The scans were examined on fractures and hemorrhages. Total body CT, optimized clinical CT of the explant and autopsy reports were compared with the micro- and diceCT scans and the results were confirmed using histological examinations. RESULTS:Sixteen potential fractures were identified in the micro-CT scans and with diceCT, nine hyperdensities were detected around potential fractures. Two previously undiscovered vital fractures were found, which were verified by histological examination. CONCLUSIONS:This research demonstrated that micro-CT can be a valuable tool to detect fractures of the HLC in forensic neck trauma cases and diceCT enables us to image the injured HLC in detail and indicate possible hemorrhages. With increasingly better stain and scan protocols and more experience gained by forensic radiologists, micro-CT could become invaluable for the examination of HLC trauma.

OBJECTIVES:Down syndrome (DS) is associated with airway abnormalities including a narrowed trachea. It is uncertain whether this narrowed trachea in DS is a consequence of deviant fetal development or an acquired disorder following endotracheal intubation after birth. This study aimed to compare the tracheal morphology in DS and non-DS fetuses using microfocus computed tomography (micro-CT). METHODS:Twenty fetal samples were obtained from the Dutch Fetal Biobank and divided into groups based on gestational age. Micro-CT images were processed to analyze tracheal length, volume, and cross-sectional area (CSA). RESULTS:Mean tracheal length and tracheal volume were similar in DS and non-DS fetuses for all gestational age groups. Mean, minimum, and maximal tracheal CSA were statistically significantly increased in the single DS fetus in the group of 21-24 weeks of gestation, but not in other gestational age groups. In 90% of all studied fetuses, the minimum tracheal CSA was located in the middle third of the trachea. CONCLUSION:Tracheal development in DS fetuses was similar to non-DS fetuses between 13 and 21 weeks of gestation. This suggests that the narrowed tracheal diameter in DS children may occur later in fetal development or results from postnatal intubation trauma. The narrowest part of the trachea is in majority of DS and non-DS fetuses the middle third. LEVEL OF EVIDENCE:3 Laryngoscope, 134:4389-4395, 2024.

Microfocus Computed Tomography (Micro-CT) is a novel method for non-destructive 3D imaging of samples, reaching microscale resolutions. While initially prominent in material sciences for small samples, micro-CT now gains significance in biological and medical studies. Here we present our utilization of micro-CT for imaging large ex-vivo human samples for anatomical and forensic research in three recent experiments and discuss the fundamentals of micro-CT imaging.For pelvic anatomical research, whole human pelvises were imaged to explore nerve anatomy around the prostate using various concentrations of buffered lugol (B-lugol). Advanced acquisition protocols were essential due to X-ray attenuation properties of the sample, which required higher energy for sufficient photon transmission.For fetal research, B-lugol stained fetuses of 20-24 gestational weeks underwent full body imaging. However, this led to challenging acquisition parameters and images of insufficient quality. Subsequent destaining yielded less dense, yet contrast-maintaining samples allowing higher quality images. Refined acquisition protocols with reduced energy improved image quality.For forensic research, explanted hyoid-larynx complexes were imaged. Micro-CT imaging showed potential in visualizing micro-fractures. The addition of B-lugol allowed for excellent soft tissue contrast and promising possibilities for forensic evaluation.In conclusion, micro-CT imaging accommodates a diversity of large ex-vivo human samples for anatomical and forensic purposes, though challenges arise with optimal soft tissue staining and acquisition protocols. We describe partial destaining as a new possibility to alleviate scanning issues to improve scan quality and highlight topics for future research. Micro-CT imaging is a promising new avenue for medical research and forensic evaluation.

BACKGROUND:Prenatal ultrasound is widely used to screen for structural anomalies before birth. While this is traditionally done in the second trimester, there is an increasing use of first-trimester ultrasound for early detection of lethal and certain severe structural anomalies. OBJECTIVES:To evaluate the diagnostic accuracy of ultrasound in detecting fetal structural anomalies before 14 and 24 weeks' gestation in low-risk and unselected pregnant women and to compare the current two main prenatal screening approaches: a single second-trimester scan (single-stage screening) and a first- and second-trimester scan combined (two-stage screening) in terms of anomaly detection before 24 weeks' gestation. SEARCH METHODS:We searched MEDLINE, EMBASE, Science Citation Index Expanded (Web of Science), Social Sciences Citation Index (Web of Science), Arts & Humanities Citation Index and Emerging Sources Citation Index (Web of Science) from 1 January 1997 to 22 July 2022. We limited our search to studies published after 1997 and excluded animal studies, reviews and case reports. No further restrictions were applied. We also screened reference lists and citing articles of each of the included studies. SELECTION CRITERIA:Studies were eligible if they included low-risk or unselected pregnant women undergoing a first- and/or second-trimester fetal anomaly scan, conducted at 11 to 14 or 18 to 24 weeks' gestation, respectively. The reference standard was detection of anomalies at birth or postmortem. DATA COLLECTION AND ANALYSIS:Two review authors independently undertook study selection, quality assessment (QUADAS-2), data extraction and evaluation of the certainty of evidence (GRADE approach). We used univariate random-effects logistic regression models for the meta-analysis of sensitivity and specificity. MAIN RESULTS:Eighty-seven studies covering 7,057,859 fetuses (including 25,202 with structural anomalies) were included. No study was deemed low risk across all QUADAS-2 domains. Main methodological concerns included risk of bias in the reference standard domain and risk of partial verification. Applicability concerns were common in studies evaluating first-trimester scans and two-stage screening in terms of patient selection due to frequent recruitment from single tertiary centres without exclusion of referrals. We reported ultrasound accuracy for fetal structural anomalies overall, by severity, affected organ system and for 46 specific anomalies. Detection rates varied widely across categories, with the highest estimates of sensitivity for thoracic and abdominal wall anomalies and the lowest for gastrointestinal anomalies across all tests. The summary sensitivity of a first-trimester scan was 37.5% for detection of structural anomalies overall (95% confidence interval (CI) 31.1 to 44.3; low-certainty evidence) and 91.3% for lethal anomalies (95% CI 83.9 to 95.5; moderate-certainty evidence), with an overall specificity of 99.9% (95% CI 99.9 to 100; low-certainty evidence). Two-stage screening had a combined sensitivity of 83.8% (95% CI 74.7 to 90.1; low-certainty evidence), while single-stage screening had a sensitivity of 50.5% (95% CI 38.5 to 62.4; very low-certainty evidence). The specificity of two-stage screening was 99.9% (95% CI 99.7 to 100; low-certainty evidence) and for single-stage screening, it was 99.8% (95% CI 99.2 to 100; moderate-certainty evidence). Indirect comparisons suggested superiority of two-stage screening across all analyses regarding sensitivity, with no significant difference in specificity. However, the certainty of the evidence is very low due to the absence of direct comparisons. AUTHORS' CONCLUSIONS:A first-trimester scan has the potential to detect lethal and certain severe anomalies with high accuracy before 14 weeks' gestation, despite its limited overall sensitivity. Conversely, two-stage screening shows high accuracy in detecting most fetal structural anomalies before 24 weeks' gestation with high sensitivity and specificity. In a hypothetical cohort of 100,000 fetuses, the first-trimester scan is expected to correctly identify 113 out of 124 fetuses with lethal anomalies (91.3%) and 665 out of 1776 fetuses with any anomaly (37.5%). However, 79 false-positive diagnoses are anticipated among 98,224 fetuses (0.08%). Two-stage screening is expected to correctly identify 1448 out of 1776 cases of structural anomalies overall (83.8%), with 118 false positives (0.1%). In contrast, single-stage screening is expected to correctly identify 896 out of 1776 cases before 24 weeks' gestation (50.5%), with 205 false-positive diagnoses (0.2%). This represents a difference of 592 fewer correct identifications and 88 more false positives compared to two-stage screening. However, it is crucial to acknowledge the uncertainty surrounding the additional benefits of two-stage versus single-stage screening, as there are no studies directly comparing them. Moreover, the evidence supporting the accuracy of first-trimester ultrasound and two-stage screening approaches primarily originates from studies conducted in single tertiary care facilities, which restricts the generalisability of the results of this meta-analysis to the broader population.

The process of vascular development is essential for shaping complex craniofacial structures. Investigating the interplay between vascular development and orofacial morphogenesis holds critical importance in clinical practice and contributes to advancing our comprehension of (vascular) developmental biology. New insights into specific vascular developmental pathways will have far-reaching implications across various medical disciplines, enhancing clinical understanding, refining surgical techniques, and elucidating the origins of congenital abnormalities. Embryonic development of the craniofacial vasculature remains, however, under-exposed in the current literature. We imaged and created 3-dimensional (D) reconstructed images of the craniofacial arterial system from two early-stage human embryonic samples. The aim of this study was to investigate the vascular development of the craniofacial region in early-stage human embryos, with a focus on understanding the interplay between vascular development and orofacial morphogenesis. Reconstructions (3-D) were generated from high-resolution diffusible iodine-based contrast-enhanced computed tomography (diceCT) images, enabling visualization of the orofacial arterial system in human embryonic samples of Carnegie stages (CS) 14 and 18 from the Dutch Fetal Biobank, corresponding to weeks 7 and 8.5 of gestation. From two human embryonic samples (ages CS 14 and 18), the vascular development of the orofacial region at two different stages of development was successfully stained with B-Lugol and imaged using a micro-computed tomography (micro-CT) scanner with resolutions of 2.5-μm and 9-μm voxel sizes, respectively. Additionally, educational 3-D reconstructions of the orofacial vascular system were generated using AMIRA 2021.2 software. Micro-CT imaging is an effective strategy for high-resolution visualization of vascular development of the orofacial region in human embryonic samples. The generated interactive 3-D educational models facilitate better understanding of the development of orofacial structures.

BACKGROUND:Visualizing (micro)vascular structures remains challenging for researchers and clinicians due to limitations in traditional radiological imaging methods. Exploring the role of vascular development in craniofacial malformations in experimental settings can enhance understanding of these processes, with the effectiveness of high-resolution imaging techniques being crucial for successful research in this field. Micro-CT imaging offers 3D microstructural insights, but requires contrast-enhancing staining agents (CESAs) for visualizing (micro)-vascular tissues, known as contrast-enhanced micro-CT (CECT). As effective contrast agents are crucial for optimal visualization, this review focuses on comparative studies investigating such agents for micro-vascular tissue imaging using micro-CT. Furthermore, we demonstrate the utilization of B-Lugol solution as a promising contrast agent for acquiring high-quality micro-CT images of (micro)vascular structures in human embryonic samples. METHOD:This scoping review followed Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocols. PubMed database provided relevant articles, screened initially by title and abstract. Inclusion and exclusion criteria defined outcomes of interest. RESULTS:From an initial search, 273 records were identified, narrowed down to 9 articles after applying our criteria. Additionally, two articles were added through citation searching. This, a total of 11 articles were incorporated in this study. CONCLUSION:This micro-CT contrast agent review underscores the need for tailored choices based on research goals. Both Barium sulfate and Iodine-based agents showing excellent results, providing high resolution (micro) vascular content, especially in ex-vivo specimens. However, careful consideration of protocols and tissue characteristics remains imperative for optimizing the effectiveness of micro-CT imaging for the study of cranio-facial vascular development.

Conclusions: Face to face confirmation of ultrasound scans by expert sonologists yields a higher accuracy though of nonstatistically significant difference compared to teleultrasound.Teleultrasound is an acceptable alternative when there is an emergent need for expert confirmation of scans and can limit exposures during a pandemic.

Recent studies of human embryos and fetuses have advanced our understanding not only of basic biology but also of health and disease, through a combination of detailed three-dimensional (3D) morphology and processes such as gene expression, cellular decision-making and differentiation, and epigenetics during the various phases of human development and growth. Large-scale research initiatives focusing on these topics have been initiated during the last decade, all of which depend on biobanks that provide high-quality images of human embryonic and fetal morphology, as well as on high-quality collections of tissue samples that are obtained and stored appropriately. In this perspective, we describe our experience in establishing the Dutch Fetal Biobank to present the framework and workflow of the biobank, provide a brief discussion of the main legal and ethical aspects involved in establishing a pre-natal tissue bank, and present the preliminary data on the first 329 donated specimens.

Over the last few years, fetal postmortem microfocus computed tomography (micro-CT) imaging has increased in popularity for both diagnostic and research purposes. Micro-CT imaging could be a substitute for autopsy, particularly in very early gestation fetuses for whom autopsy can be technically challenging and is often unaccepted by parents. This article provides an overview of the latest research in fetal postmortem micro-CT imaging with a focus on diagnostic accuracy, endovascular staining approaches, placental studies and the reversibility of staining. It also discusses new methods that could prove helpful for micro-CT of larger fetuses. While more research is needed, contrast-enhanced micro-CT has the potential to become a suitable alternative to fetal autopsy. Further research using this novel imaging tool could yield wider applications, such as its practise in imaging rare museum specimens.