Defective airway intraflagellar transport underlies a combined motile and primary ciliopathy syndrome caused by IFT74 mutations.

Ciliopathies are inherited disorders caused by defective cilia. Mutations affecting motile cilia usually cause the chronic muco-obstructive sinopulmonary disease primary ciliary dyskinesia (PCD) and are associated with laterality defects, while a broad spectrum of early developmental as well as degenerative syndromes arise from mutations affecting signalling of primary (non-motile) cilia. Cilia assembly and functioning requires intraflagellar transport (IFT) of cargos assisted by IFT-B and IFT-A adaptor complexes. Within IFT-B, the N-termini of partner proteins IFT74 and IFT81 govern tubulin transport to build the ciliary microtubular cytoskeleton. We detected a homozygous 3-kb intragenic IFT74 deletion removing the exon 2 initiation codon and 40 N-terminal amino acids in two affected siblings. Both had clinical features of PCD with bronchiectasis, but no laterality defects. They also had retinal dysplasia and abnormal bone growth, with a narrowed thorax and short ribs, shortened long bones and digits, and abnormal skull shape. This resembles short-rib thoracic dysplasia, a skeletal ciliopathy previously linked to IFT defects in primary cilia, not motile cilia. Ciliated nasal epithelial cells collected from affected individuals had reduced numbers of shortened motile cilia with disarranged microtubules, some misorientation of the basal feet, and disrupted cilia structural and IFT protein distributions. No full-length IFT74 was expressed, only truncated forms that were consistent with N-terminal deletion and inframe translation from downstream initiation codons. In affinity purification mass spectrometry, exon 2-deleted IFT74 initiated from the nearest inframe downstream methionine 41 still interacts as part of the IFT-B complex, but only with reduced interaction levels and not with all its usual IFT-B partners. We propose that this is a hypomorphic mutation with some residual protein function retained, which gives rise to a primary skeletal ciliopathy combined with defective motile cilia and PCD.

Ultrastructural cilia defects in multi-ciliated uterine glandular epithelial cells from women with reproductive failure.

In brief: The causes of subfertility and recurrent pregnancy loss are often unclear. This study shows that endometrial gland cilia from women with subfertility have ultrastructural defects. Abstract: Endometrial glands secrete products into the endometrium and are necessary for embryo implantation and successful pregnancy. However, structural and functional abnormalities in endometrial gland cilia from women with reproductive failure remain poorly understood. This was a cross-sectional study where endometrial biopsies were collected at days 19-23 of the menstrual cycle from women with unexplained recurrent pregnancy loss (n = 15), unexplained subfertility (n = 11) or from egg donor control participants (n = 10). Endometrial gland cilia ultrastructure was imaged by transmission electron microscopy and cilia defects assessed by an electron-microscopist from a national primary ciliary dyskinesia diagnostic centre. Endometrial glands were isolated, and the cilia beat frequency recorded by high speed video. Subfertile women have proportionately lower ultrastructurally normal cilia (P < 0.05); higher frequency of absent dynamin arms (P < 0.01) or inner arm defects (P < 0.01) and lower cilia beat frequency (P < 0.05). The mechanisms underlying these obversions have yet to be determined. Recent studies have identified cilia related gene expression changes associated with reproductive failure and this study adds to the growing body of literature revealing structural and functional changes. The observation that cilia defects occurred at a higher frequency in endometrial glands of subfertile women raises the question of its mechanistic role in implantation.

Convergently evolved placental villi show multiscale structural adaptations to differential placental invasiveness.

Despite having a single evolutionary origin and conserved function, the mammalian placenta exhibits radical structural diversity. The evolutionary drivers and functional consequences of placental structural diversity are poorly understood. Humans and equids both display treelike placental villi, however these villi evolved independently and exhibit starkly different levels of invasiveness into maternal tissue (i.e. the number of maternal tissue layers between placental tissue and maternal blood). The villi in these species therefore serve as a compelling evolutionary case study to explore whether placentas have developed structural adaptations to respond to the challenge of reduced nutrient availability in less invasive placentas. Here, we use three-dimensional X-ray microfocus computed tomography and electron microscopy to quantitatively evaluate key structures involved in exchange in human and equid placental villi. We find that equid villi have a higher surface area to volume ratio and deeper trophoblastic vessel indentation than human villi. Using illustrative computational models, we propose that these structural adaptations have evolved in equids to boost nutrient transfer to compensate for reduced invasiveness into maternal tissue. We discuss these findings in relation to the 'maternal-fetal conflict hypothesis' of placental evolution.

2D vs. 3D Evaluation of Osteocyte Lacunae - Methodological Approaches, Recommended Parameters, and Challenges: A Narrative Review by the European Calcified Tissue Society (ECTS).

PURPOSE OF REVIEW: Quantification of the morphology of osteocyte lacunae has become a powerful tool to investigate bone metabolism, pathologies and aging. This review will provide a brief overview of 2D and 3D imaging methods for the determination of lacunar shape, orientation, density, and volume. Deviations between 2D-based and 3D-based lacunar volume estimations are often not sufficiently addressed and may give rise to contradictory findings. Thus, the systematic error arising from 2D-based estimations of lacunar volume will be discussed, and an alternative calculation proposed. Further, standardized morphological parameters and best practices for sampling and segmentation are suggested. RECENT FINDINGS: We quantified the errors in reported estimation methods of lacunar volume based on 2D cross-sections, which increase with variations in lacunar orientation and histological cutting plane. The estimations of lacunar volume based on common practice in 2D imaging methods resulted in an underestimation of lacunar volume of up to 85% compared to actual lacunar volume in an artificial dataset. For a representative estimation of lacunar size and morphology based on 2D images, at least 400 lacunae should be assessed per sample.

Clinical utility of NGS diagnosis and disease stratification in a multiethnic primary ciliary dyskinesia cohort.

BACKGROUND: Primary ciliary dyskinesia (PCD), a genetically heterogeneous condition enriched in some consanguineous populations, results from recessive mutations affecting cilia biogenesis and motility. Currently, diagnosis requires multiple expert tests. METHODS: The diagnostic utility of multigene panel next-generation sequencing (NGS) was evaluated in 161 unrelated families from multiple population ancestries. RESULTS: Most (82%) families had affected individuals with biallelic or hemizygous (75%) or single (7%) pathogenic causal alleles in known PCD genes. Loss-of-function alleles dominate (73% frameshift, stop-gain, splice site), most (58%) being homozygous, even in non-consanguineous families. Although 57% (88) of the total 155 diagnostic disease variants were novel, recurrent mutations and mutated genes were detected. These differed markedly between white European (52% of families carry DNAH5 or DNAH11 mutations), Arab (42% of families carry CCDC39 or CCDC40 mutations) and South Asian (single LRRC6 or CCDC103 mutations carried in 36% of families) patients, revealing a striking genetic stratification according to population of origin in PCD. Genetics facilitated successful diagnosis of 81% of families with normal or inconclusive ultrastructure and 67% missing prior ultrastructure results. CONCLUSIONS: This study shows the added value of high-throughput targeted NGS in expediting PCD diagnosis. Therefore, there is potential significant patient benefit in wider and/or earlier implementation of genetic screening.

Occupational Localized Cutaneous Argyria With Pseudo-Ochronosis in a Jeweler.

ABSTRACT: A case of localized argyria in a 36-year-old female jeweler is described who presented with 2 discrete and asymptomatic bluish-black pigmented macules on the pulp of her left middle finger. A skin biopsy from both lesions demonstrated deposition of brown/black pigmented granules along the basement membrane zone of eccrine glands, blood vessels, nerves, and the dermo-epidermal junction fully in keeping with silver deposition. In addition, there was yellow-brown deposition seen within the interstitial dermis mimicking an early form of ochronosis, so called "pseudo-ochronosis." This latter feature is rarely described in cases of argyria. Transmission electron microscopy and energy dispersive x-ray spectroscopy confirmed the presence of electron dense particles up to 150 nm in diameter and the presence of silver, respectively. On further questioning, the patient had a history of localized and chronic exposure to silver, which specifically involved holding and manipulating silver wires and rings over the left middle finger. This case highlights an unusual and rare presentation of localized argyria in a jeweler. In addition, our case showed preferential silver deposition on dermal elastic fibers which has not been previously described in the literature.

International consensus guideline for reporting transmission electron microscopy results in the diagnosis of primary ciliary dyskinesia (BEAT PCD TEM Criteria).

Primary ciliary dyskinesia (PCD) is a heterogeneous genetic condition. European and North American diagnostic guidelines recommend transmission electron microscopy (TEM) as one of a combination of tests to confirm a diagnosis. However, there is no definition of what constitutes a defect or consensus on reporting terminology. The aim of this project was to provide an internationally agreed ultrastructural classification for PCD diagnosis by TEM.A consensus guideline was developed by PCD electron microscopy experts representing 18 centres in 14 countries. An initial meeting and discussion were followed by a Delphi consensus process. The agreed guideline was then tested, modified and retested through exchange of samples and electron micrographs between the 18 diagnostic centres.The final guideline a) provides agreed terminology and a definition of Class 1 defects which are diagnostic for PCD; b) identifies Class 2 defects which can indicate a diagnosis of PCD in combination with other supporting evidence; c) describes features which should be included in a ciliary ultrastructure report to assist multidisciplinary diagnosis of PCD; and d) defines adequacy of a diagnostic sample.This tested and externally validated statement provides a clear guideline for the diagnosis of PCD by TEM which can be used to standardise diagnosis internationally.

Serial block-face scanning electron microscopy reveals novel intercellular connections in human term placental microvasculature.

The placental microvasculature is a conduit for fetal blood allowing solute exchange between the mother and the fetus. Serial block-face scanning electron microscopy (SBF SEM) allows ultrastructure to be viewed in three dimensions and provides a new perspective on placental anatomy. This study used SBF SEM to study endothelial cells within the human placental microvasculature from uncomplicated pregnancies. Term human placental villi were aldehyde-fixed and processed for imaging by SBF SEM. Manual segmentation was carried out on a terminal villous capillary and an intermediate villous arteriole and venule. Twenty-seven SBF SEM stacks from terminal villi were analysed using stereological approaches to determine the volumes of microvascular components and the proportions of pericyte coverage. SBF SEM analysis of capillary endothelial cells revealed the presence of interendothelial protrusions (IEPs) originating from the donor cell at the endothelial junction and forming deep thin projections up to 7 µm into the adjacent endothelial cells. IEP density was estimated to be in the order of 35 million cm-3 placental tissue. Pericytes cover 15% of the fetal capillary surface area in terminal villi. In comparison, the cytotrophoblast covered 24% of the syncytiotrophoblast basal membrane. A trans-endothelial channel was observed in a region of the vasculo-syncytial capillary. Pericyte coverage was extensive in both arteriole and venule. Three-dimensional imaging of the placental microvasculature identified novel ultrastructural features and provided an insight into factors that may influence capillary permeability and placental function. We hypothesise that the IEPs may allow mechanosensing between adjacent endothelial cells to assist in the maintenance of vessel integrity. The numbers of endothelial junctions, the presence of trans-endothelial channels and the extent of pericyte coverage all provide an insight into the factors determining capillary permeability.

3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina.

The retinal pigment epithelium (RPE) is located between the neuroretina and the choroid, and plays a critical role in vision. RPE cells internalise outer segments (OS) from overlying photoreceptors in the daily photoreceptor renewal. Changes to RPE structure are linked with age and retinopathy, which has been described in the past by conventional 2D electron microscopy. We used serial block face scanning electron microscopy (SBF-SEM) to reconstruct RPE cells from the central mouse retina. Three-dimensional-reconstructed OS revealed the RPE to support large numbers of photoreceptors (90-216 per RPE cell). Larger bi-nucleate RPE maintained more photoreceptors, although their cytoplasmic volume was comparable to smaller mono-nucleate RPE supporting fewer photoreceptors. Scrutiny of RPE microvilli and interdigitating OS revealed the angle and surface area of contact between RPE and photoreceptors. Bi-nucleate RPE contained more mitochondria compared to mono-nucleate RPE. Furthermore, bi-nucleate cells contained larger sub-RPE spaces, supporting a likely association with disease. Use of perfusion-fixed tissues ensured the highest possible standard of preservation, providing novel insights into the 3D RPE architecture and changes linked with retinopathy. This study serves as a benchmark for comparing retinal tissues from donor eyes with age-related macular degeneration (AMD) and other retinopathies.

Risk factors for situs defects and congenital heart disease in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is associated with abnormal organ positioning (situs) and congenital heart disease (CHD). This study investigated genotype-phenotype associations in PCD to facilitate risk predictions for cardiac and laterality defects. This retrospective cohort study of 389 UK patients with PCD found 51% had abnormal situs and 25% had CHD and/or laterality defects other than situs inversus totalis. Patients with biallelic mutations in a subset of nine PCD genes had normal situs. Patients with consanguineous parents had higher odds of situs abnormalities than patients with non-consanguineous parents. Patients with abnormal situs had higher odds of CHD and/or laterality defects.

High prevalence of CCDC103 p.His154Pro mutation causing primary ciliary dyskinesia disrupts protein oligomerisation and is associated with normal diagnostic investigations.

RATIONALE: Primary ciliary dyskinesia is a genetically heterogeneous inherited condition characterised by progressive lung disease arising from abnormal cilia function. Approximately half of patients have situs inversus. The estimated prevalence of primary ciliary dyskinesia in the UK South Asian population is 1:2265. Early, accurate diagnosis is key to implementing appropriate management but clinical diagnostic tests can be equivocal. OBJECTIVES: To determine the importance of genetic screening for primary ciliary dyskinesia in a UK South Asian population with a typical clinical phenotype, where standard testing is inconclusive. METHODS: Next-generation sequencing was used to screen 86 South Asian patients who had a clinical history consistent with primary ciliary dyskinesia. The effect of a CCDC103 p.His154Pro missense variant compared with other dynein arm-associated gene mutations on diagnostic/phenotypic variability was tested. CCDC103 p.His154Pro variant pathogenicity was assessed by oligomerisation assay. RESULTS: Sixteen of 86 (19%) patients carried a homozygous CCDC103 p.His154Pro mutation which was found to disrupt protein oligomerisation. Variable diagnostic test results were obtained including normal nasal nitric oxide levels, normal ciliary beat pattern and frequency and a spectrum of partial and normal dynein arm retention. Fifteen (94%) patients or their sibling(s) had situs inversus suggesting CCDC103 p.His154Pro patients without situs inversus are missed. CONCLUSIONS: The CCDC103 p.His154Pro mutation is more prevalent than previously thought in the South Asian community and causes primary ciliary dyskinesia that can be difficult to diagnose using pathology-based clinical tests. Genetic testing is critical when there is a strong clinical phenotype with inconclusive standard diagnostic tests.

Serial block-face scanning electron microscopy of erythrocytes protruding through the human placental syncytiotrophoblast.

The syncytiotrophoblast forms a continuous barrier between the maternal and fetal circulations. Here we present a serial block-face scanning electron microscopy (SBFSEM) study, based on a single image stack, showing pooling of fetal blood underneath a region of stretched syncytiotrophoblast that has become detached from the basement membrane. Erythrocytes are protruding from discrete holes in the syncytiotrophoblast suggesting that, under specific circumstances, the syncytiotrophoblast may be permeable to fetal cells. This observation represents a pathological process but it poses questions about the physical properties and permeability of the syncytiotrophoblast and may represent an early stage in the formation of fibrin deposits in areas of syncytial denudation. This study also illustrates how the 3D images generated by SBFSEM allow the interpretation of structures that could not be understood from a single histological section.

Accuracy of diagnostic testing in primary ciliary dyskinesia.

Diagnosis of primary ciliary dyskinesia (PCD) lacks a "gold standard" test and is therefore based on combinations of tests including nasal nitric oxide (nNO), high-speed video microscopy analysis (HSVMA), genotyping and transmission electron microscopy (TEM). There are few published data on the accuracy of this approach.Using prospectively collected data from 654 consecutive patients referred for PCD diagnostics we calculated sensitivity and specificity for individual and combination testing strategies. Not all patients underwent all tests.HSVMA had excellent sensitivity and specificity (100% and 93%, respectively). TEM was 100% specific, but 21% of PCD patients had normal ultrastructure. nNO (30 nL·min(-1) cut-off) had good sensitivity and specificity (91% and 96%, respectively). Simultaneous testing using HSVMA and TEM was 100% sensitive and 92% specific.In conclusion, combination testing was found to be a highly accurate approach for diagnosing PCD. HSVMA alone has excellent accuracy, but requires significant expertise, and repeated sampling or cell culture is often needed. TEM alone is specific but misses 21% of cases. nNO (≤30 nL·min(-1)) contributes well to the diagnostic process. In isolation nNO screening at this cut-off would miss ∼10% of cases, but in combination with HSVMA could reduce unnecessary further testing. Standardisation of testing between centres is a future priority.

Diagnostic Methods in Primary Ciliary Dyskinesia.

Diagnosing primary ciliary dyskinesia is difficult. With no reference standard, a combination of tests is needed; most tests require expensive equipment and specialist scientists. We review the advances in diagnostic testing over the past hundred years, with emphasis on recent advances. We particularly focus on use of high-speed video analysis, transmission electron microscopy, nasal nitric oxide and genetic testing. We discuss the international efforts that are in place to advance the evidence base for diagnostic tests.

A case report of primary ciliary dyskinesia, laterality defects and developmental delay caused by the co-existence of a single gene and chromosome disorder.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder characterised by abnormal ciliary motion and impaired mucociliary clearance, leading to recurrent respiratory infections, sinusitis, otitis media and male infertility. Some patients also have laterality defects. We recently reported the identification of three disease-causing PCD genes in the Irish Traveller population; RSPH4A, DYX1C1 and CCNO. We have since assessed an additional Irish Traveller family with a complex phenotype involving PCD who did not have any of the previously identified PCD mutations. CASE PRESENTATION: In this study we report on a family with three children with PCD and various laterality defects. In addition, one child (V:1) has mild-to-moderate developmental delay and one child has speech delay (V:2). Developmental delay is not usually associated with PCD and is likely to be caused by an additional genetic abnormality. Transmission electron microscopy showed variable inner and outer dynein arm defects. Exome sequencing identified a homozygous missense variant in CCDC103 (c.461A > C; p.His154Pro) as the most likely cause of the PCD and laterality defects in this family. However, as mutation in CCDC103 would not account for the developmental delay, array comparative genomic hybridisation was undertaken and identified a maternally inherited gain of ~1.6 Mb (chr17:34,611,352-36,248,918). Gains at this locus are associated with 17q12 duplication syndrome which includes speech and language delay. CONCLUSION: We report on a variable and complex phenotype caused by the co-inheritance of a single gene mutation in CCDC103 and a microduplication at 17q12, both on chromosome 17. The co-existence of a single gene and chromosome disorder is unusual but accounts for the spectrum of clinical features in this family. In addition, our study brings the total number of PCD genes in the Irish Traveller population to four and we suspect additional PCD genes are yet to be identified. Although, on a global scale, PCD is associated with extensive genetic heterogeneity, finding such a high number of causative PCD genes within the relatively small Irish Traveller population was unexpected.

A Novel Sandwich Method for Serial Block Face SEM Imaging of Airway Multiciliated Epithelium.

Volume electron microscopy technologies such as serial block face scanning electron microscopy (SBF-SEM) allow the characterization of tissue organization and cellular content in three dimensions at nanoscale resolution. Here, we describe the procedure to process and image an air-liquid interface culture of human or mouse airway epithelial cells for visualization of the multiciliated epithelium by SBF-SEM in vertical or horizontal cross section.

Mutations in CCDC39 and CCDC40 are the major cause of primary ciliary dyskinesia with axonemal disorganization and absent inner dynein arms.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed "radial spoke defect." We sequenced CCDC39 and CCDC40 in 54 "radial spoke defect" families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice, and frameshift predicting early protein truncation, which suggests this defect is caused by "null" alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganization and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as "IDA and microtubular disorganisation defect," rather than "radial spoke defect."

Correlative multiscale microCT-SBF-SEM imaging of resin-embedded tissue.

Three-dimensional biological microscopy presents a trade-off between spatial resolution and field of view. Correlative approaches applying multiple imaging techniques to the same sample can therefore mitigate against these trade-offs. Here, we present a workflow for correlative microscopic X-ray microfocus computed tomography (microCT) and serial block face scanning electron microscopy (SBF-SEM) imaging of resin-embedded tissue, using mammalian placental tissue samples as an example. This correlative X-ray and electron microscopy (CXEM) workflow allows users to image the same sample at multiple resolutions, and target the region of interest (ROI) for SBF-SEM based on microCT. We detail the protocols associated with this workflow and demonstrate its application in multiscale imaging of horse placental villi and ROI selection in the labyrinthine zone of a mouse placenta. These examples demonstrate how the protocol may need to be adapted for tissues with different densities.

Subconjunctival Ocular Argyrosis following Treatment with Ruthenium 106 Brachytherapy for Choroidal Melanoma.

Introduction: Ruthenium-106 (Ru-106) brachytherapy is one of the commonest eye-sparing treatments for choroidal melanoma. These patients require long-term surveillance of the treated tumour remnant to ensure there is no local recurrence. New or progressive pigmented lesions in treated eyes are often regarded as suspicious - especially if there are concerns of extra-scleral extension. Case Presentations: We present two cases of posterior choroidal melanoma treated five and 10 years previously with Ru-106. Both cases developed subconjunctival dark/black lesions on the anterior surface of the eye in the quadrant of the conjunctival peritomy during Ru-106 treatment. Both had similar findings on histopathology: black, non-organic, particulate foreign material of varying confluence deposited on elastin and collagen fibres. Energy dispersive X-ray microanalysis confirmed the material contained silver. Discussion: The Ru-106 applicator consists of a radioactive core of Ru-106 encapsulated within pure silver as a radiation shield. During surgical insertion, stainless steel suture needles and forceps can occasionally scratch the applicator's silver eyelets and scatter microscopic particles of elemental silver into the operative field. These particles were likely deposited within the subconjunctival tissues of these patients during brachytherapy administration, leading to localised ocular argyrosis. Iatrogenic ocular argyrosis should be considered in the differential diagnosis of new pigmented lesions in patients treated with Ru-106 brachytherapy. This study is the first to unequivocally identify the cause of some post-brachytherapy ocular surface pigmentation as caused by silver.

A high-throughput 3D X-ray histology facility for biomedical research and preclinical applications.

Background: The University of Southampton, in collaboration with the University Hospital Southampton (UHS) NHS Foundation Trust and industrial partners, has been at the forefront of developing three-dimensional (3D) imaging workflows using X-ray microfocus computed tomography (µCT) -based technology. This article presents the outcomes of these endeavours and highlights the distinctive characteristics of a µCT facility tailored explicitly for 3D X-ray Histology, with a primary focus on applications in biomedical research and preclinical and clinical studies. Methods: The UHS houses a unique 3D X-ray Histology (XRH) facility, offering a range of services to national and international clients. The facility employs specialised µCT equipment explicitly designed for histology applications, allowing whole-block XRH imaging of formalin-fixed and paraffin-embedded tissue specimens. It also enables correlative imaging by combining µCT imaging with other microscopy techniques, such as immunohistochemistry (IHC) and serial block-face scanning electron microscopy, as well as data visualisation, image quantification, and bespoke analysis. Results: Over the past seven years, the XRH facility has successfully completed over 120 projects in collaboration with researchers from 60 affiliations, resulting in numerous published manuscripts and conference proceedings. The facility has streamlined the µCT imaging process, improving productivity and enabling efficient acquisition of 3D datasets. Discussion & Conclusions: The 3D X-ray Histology (XRH) facility at UHS is a pioneering platform in the field of histology and biomedical imaging. To the best of our knowledge, it stands out as the world's first dedicated XRH facility, encompassing every aspect of the imaging process, from user support to data generation, analysis, training, archiving, and metadata generation. This article serves as a comprehensive guide for establishing similar XRH facilities, covering key aspects of facility setup and operation. Researchers and institutions interested in developing state-of-the-art histology and imaging facilities can utilise this resource to explore new frontiers in their research and discoveries.