Enhancing SEM positioning precision with a LEGO®-based sample fitting system.

Scanning electron microscopy (SEM) is a precious tool in materials science and morphology sciences, enabling detailed examination of materials at the nanoscale. However, precise and accurate sample repositioning during different observation sessions remains a significant challenge, impacting the quality and repeatability of SEM analyses. This study aimed to develop and evaluate a LEGO®-based sample positioning system for SEM analysis. The system was designed to consistently identify and align features across multiple repositioning cycles, maintain accurate positioning along the z-axis, minimize distortion, and provide repeatable and reliable results. The results indicated a high degree of precision and accuracy in the repositioning process, as evidenced by the minimal displacements, deviations in scaling and shearing, and the highly significant results (p < 0.001) obtained from the analysis of absolute translations and rotations. Moreover, the analyses were consistently replicated across six repetitions, underscoring the reliability of the observed results. While the findings suggest that the LEGO-based sample positioning system is promising for enhancing SEM analyses' quality and repeatability, further studies are needed to optimize the system's design and evaluate its performance in different SEM applications. Ultimately, this study contributes to the ongoing efforts to develop cost-effective, customizable, and accurate solutions for sample positioning in SEM, contributing to the advancement of materials science research and all SEM analysis requiring overtime observations of the same sample. RESEARCH HIGHLIGHTS: This study focused on the development and evaluation of a novel LEGO-based sample positioning system specifically designed for SEM analysis. One of the standout features of this system is its ability to consistently identify and align features across multiple repositioning cycles, showcasing its precision and reliability. To further understand the mechanical aspects of the SEM stage, we employed the Rambold Kontroll comparator, which provided a baseline understanding of its mechanical tolerance. The registration process results were particularly noteworthy, as they revealed high accuracy with minimal displacements. Furthermore, the consistent outcomes observed across multiple repetitions emphasize the reliability and robustness of the methods we employed in this research.

Investigating the interfaces of the epiphyseal plate: An integrated approach of histochemistry, microtomography and SEM.

We investigated the interfaces of the epiphyseal plate with over- and underlying bone segments using an integrated approach of histochemistry, microtomography and scanning electron microscopy (SEM) to overcome the inherent limitations of sections-based techniques. Microtomography was able to provide an unobstructed, frontal view of large expanses of the two bone surfaces facing the growth plate, while SEM observation after removal of the soft matrix granted an equally unhindered access with a higher resolution. The two interfaces appeared widely dissimilar. On the diaphyseal side the hypertrophic chondrocytes were arranged in tall columns packed in a sort of compact palisade; the interposed extracellular matrix was actively calcifying into a thick mineralized crust growing towards the epiphysis. Behind the mineralization front, histochemical data revealed a number of surviving cartilage islets which were being slowly remodelled into bone. In contrast, the epiphyseal side of the cartilage consisted of a relatively quiescent reserve zone whose mineralization was marginal in amount and discontinuous in extension; the epiphyseal bone consisted of a loose trabecular meshwork, with ample vascular spaces opening directly into the non-mineralized cartilage. On both sides the calcification process took place through the formation of spheroidal bodies 1-2 µm wide which gradually grew by apposition and coalesced into a solid mass, in a way distinctly different from that of bone and other calcified tissues.

The chondro-osseous junction of articular cartilage.

In the synovial joints the transition between the soft articular cartilage and the subchondral bone is mediated by a layer of calcified cartilage of structural and mechanical characteristics closer to those of bone. This layer, buried in the depth of articular cartilage, is not directly accessible and is mostly visualized in histological sections of decalcified tissue, where it appears as a darker strip in contact with the subchondral bone. In this study conventional histology and scanning electron microscopy (SEM) with secondary electron imaging (SE) or backscattered electron imaging (BSE) were used to discriminate the calcified and the uncalcified cartilage in high resolution on native, untreated tissue as well as in deproteinated or demineralized tissue. This approach evidenced a high heterogeneity of the calcified layer of articular cartilage. High resolution pictures revealed that the mineralization process originates by progressive accretion and confluence of individual, small mineral clusters, in a very different way from other hard tissues such as bone, dentin and mineralized tendons. Finally, selective removal of the soft matrix by thermal treatment allowed for the first time a face-on, unrestricted 3D view of the mineralization front.

HA PEGylated Filler in Association with an Infrared Energy Device for the Treatment of Facial Skin Aging: 150 Day Follow-Up Data Report.

BACKGROUND: The face is the area most exposed to the normal course of skin aging, both intrinsically and extrinsically. The aim of the study was to evaluate the cellular and clinical response of a therapeutic protocol aimed at countering facial skin aging. MATERIALS AND METHODS: Twenty female patients with facial skin laxity and photodamage underwent combined therapy including mesotherapy using non-cross-linked hyaluronic acid with calcium hydroxyapatite and an infrared energy-based device treatment with subsequent implementation of PEG-cross-linked hyaluronic acid soft tissue fillers. To evaluate the benefits, patients underwent histological, immunological, and biomechanical evaluations before the treatment and at 21 and 150 days after the treatment. RESULTS: The histological results at 21 days and 150 days after the procedure showed an increase in the number of fibroblasts and angiogenesis. As for the immunological aspect, it was shown that the treatment has an immunomodulating action, avoiding the activation of CD4 and CD8 cells. Biomechanical data showed that, at 150 days after treatment, the average changes in skin elasticity increased by 72% and the skin hydration increased by 49%. CONCLUSIONS: A combination of an infrared energy-based device treatment with both non-cross-linked hyaluronic acid and novel PEG-cross-linked hyaluronic acid leads to numerous positive cutaneous changes after histological, immunological, and biomechanical evaluations.

In Vitro Hair Growth Promoting Effect of a Noncrosslinked Hyaluronic Acid in Human Dermal Papilla Cells.

Dermal papilla cells (DPCs) are a source of nutrients and growth factors, which support the proliferation and growth of keratinocytes as well as promoting the induction of new hair follicles and maintenance of hair growth. The protection from reactive oxygen species (ROS) and the promotion of angiogenesis are considered two of the basal mechanisms to preserve the growth of the hair follicle. In this study, a noncrosslinked hyaluronic acid (HA) filler (HYDRO DELUXE BIO, Matex Lab S.p.A.) containing several amino acids was tested with in vitro assays on human follicle dermal papilla cells (HFDPCs). The experiments were carried out to investigate the possible protection against oxidative stress and the ability to increase the vascular endothelial growth factor (VEGF) release. The results demonstrated the restoration of cell viability against UVB-induced cytotoxicity and an increase in the VEGF secretion. These data demonstrate the capability of the product to modulate human dermal papilla cells, suggesting a future use in mesotherapy, a minimally invasive local intradermal therapy (LIT), after further clinical investigations.

Toward Physicochemical and Rheological Characterization of Different Injectable Hyaluronic Acid Dermal Fillers Cross-Linked with Polyethylene Glycol Diglycidyl Ether.

(1) Background: Injectable hyaluronic acid (HA) dermal fillers are used to restore volume, hydration and skin tone in aesthetic medicine. HA fillers differ from each other due to their cross-linking technologies, with the aim to increase mechanical and biological activities. One of the most recent and promising cross-linkers is polyethylene glycol diglycidyl ether (PEGDE), used by the company Matex Lab S.p.A., (Brindisi, Italy) to create the HA dermal filler PEGDE family. Over the last few years, several studies have been performed to investigate the biocompatibility and biodegradability of these formulations, but little information is available regarding their matrix structure, rheological and physicochemical properties related to their cross-linking technologies, the HA content or the degree of cross-linking. (2) Methods: Seven different injectable HA hydrogels were subjected to optical microscopic examination, cohesivity evaluation and rheological characterization in order to investigate their behavior. (3) Results: The analyzed cross-linked dermal fillers showed a fibrous "spiderweb-like" matrix structure, with each medical device presenting different and peculiar rheological features. Except for HA non cross-linked hydrogel 18 mg/mL, all showed an elastic and cohesive profile. (4) Conclusions: The comparative analysis with other literature works makes a preliminary characterization of these injectable medical devices possible.

Photon emission and changes in fluorescent properties of bone after laser irradiation.

Laser scalpels used in medical surgery concentrate light energy, heating the tissues. Recently, we reported thermoluminescence emission from laser-treated soft tissues. Here we investigated the thermo-optical effects caused by a laser operating at 808 nm on animal bones (beef ribs) through luminescence and fluorescence imaging, thermal imaging and scanning electron microscopy. Laser-induced artificial lesions emitted luminescence peaking around 650 nm, with a half-life of almost 1 hour. As concerns fluorescence, 24 hours after laser treatment we observed an increase of the emission and a shift from 500 (untreated) to 580 nm (treated). Recrystallization observed by SEM indicates that the temperature in the artificial lesions is over 600°C. We can conclude that laser treatment induces specific luminescent and fluorescent emissions due to heating of the bone and modification of its components. Monitoring these emissions could help prevent tissue overheating and its potential damages during laser-assisted medical procedures.

In Vitro Evaluation of the Effect of a Not Cross-Linked Hyaluronic Acid Hydrogel on Human Keratinocytes for Mesotherapy.

The multicomponent preparations for mesotherapy are based on the principle that skin and hair aging can be prevented by supplying the fundamental substrates for correct cellular functioning, such as nucleotides, vitamins, amino acids, and biomolecules including hyaluronic acid (HA) that promote skin hydration and several biological activities. The study provides evidence for the application of HYDRO DELUXE BIO (Matex Lab S.p.A), a biocompatible hydrogel containing not cross-linked HA, for the treatment of the scalp's skin by mesotherapy. Using an in vitro model of immortalized human keratinocytes, we studied markers involved in hair aging prevention and growth, such as inflammatory markers, angiogenesis, and oxidative damage. HYDRO DELUXE BIO showed high biocompatibility and the ability to significantly reduce the expression of the inflammation marker interleukin (IL)-8 in Tumor Necrosis Factor (TNF)-stimulated cells. Then, we evaluated angiogenesis, a pivotal event during hair growth, measuring the Vascular Endothelial Growth Factor (VEGF) expression that resulted to be significantly increased in treated cells, suggesting a pro-angiogenetic capability. A protective activity against the oxidative stress agent was showed, increasing the survival rate in treated cells. Concluding, HYDRO DELUXE BIO is suitable for treatment by mesotherapy of the scalp's skin as it modulates the expression levels of markers involved in the biorevitalization of the hair follicle.

Slippery when wet: The free surface of the articular cartilage.

The free surface of the articular cartilage must withstand compressive and shearing forces, maintain a low friction coefficient and allow oxygen and metabolites to reach the underlying matrix. In many ways it is critical to the physiology of the whole tissue and its disruption always involves deep pathological alterations and loss of the joint integrity. Being very difficult to image with section-based conventional techniques, it was often described by previous research in conflicting terms or entirely overlooked. High-magnification face-on observations with high resolution scanning electron microscopy and with scanning probe microscopy revealed a very thin, delicate superficial layer rich in glycoconjugates, which may explain the very low friction coefficient of the tissue but which was very easily altered and/or dissolved in the preparation. Beneath this superficial sheet lies a thicker coat of thin, highly uniform, slightly wavy collagen fibrils lying parallel to the surface and mutually interconnected by a huge number of interfibrillar glycosaminoglycan bridges. These bridges and the collagen fibrils form an extended reticular structure able to redistribute tensile and compressive stress across a larger area of the surface and hence a greater volume of tissue.

Combined laser assisted treatment for permanent hair removal for skin types I-V with Alexandrite 755 nm and ND:YAG 1064 nm lasers.

This retrospective cohort study regarding hair removal procedures considers all the data collected in three different clinical centers from 2017 to 2019. The device used to perform the treatments was composed of an Alexandrite 755 nm and an Nd:YAG 1064 nm laser, that delivered both wavelengths in a blended simultaneous emission (Thunder MT, Quanta System, Samarate, Italy). The improvement evaluated after five sessions of treatment by an external referee was: 83.0% for armpits, 82.1% for the bikini line, 82.2% for legs, 79.6% for thorax, and 81.6% for the back. The collected temporary skin reaction data reported in this study were all acceptable and transitory resolving in less than 1 week. The level of erythema and perifollicular edema were all signs of the reached treatment endpoint. First degree burns, hyper, and hypopigmentation were also reported in a few cases but these, all resolved before the follow-up visit without any permanent skin effects. No adverse effects were thus reported to have happened. This retrospective study demonstrates the efficacy of combining Alexandrite and Nd:YAG lasers in a mixed modality with simultaneous emission. This technology permitted to treat patients with skin types I-V without any reported permanent side effects and with a high pain tolerability compared to the use of Nd:YAG in single mode.

Skin vascular lesions: A new therapeutic option with sequential laser-assisted technique.

Treatment of skin unaesthetic vascular lesions is still suboptimal. In this article, we present a multicenter study evaluating the efficacy and safety of the sequential 755 nm Alexandrite and 1064 nm ND:YAG lasers for the treatment of acquired and congenital vascular lesions of the skin. A total of 171 patients were included in the study (141 female and 30 male), median age 38, skin type from I to IV. Numbers of patients categorized for treated disease: 22 spider angiomas, 67 facial telangiectasia/spider veins, 58 leg telangiectasia/reticular leg veins, 24 port wine stains (PWS). Of those lesions 30.7% was on legs, 63.7% on face (6.7% on forehead, 10.1% on temples, 21.7% on cheeks, 25.2% on nose, 1.7% on chin) and 5.6% on chest. Spider angiomas required from 1 to 3 treatments. Facial telangiectasias had been treated from 1 to 4 times. Leg telangiectasias needed from 2 to 6 treatment sessions. Regarding PWS, it takes from 6 to 9 laser treatment to reach the clearance of the lesion. All the vascular disorders treated showed marked improvement while side effects were limited to low pain sensation and transitory fenomena. This sequential treatment is an effective, safe, and new approach for unaesthetic skin vascular lesions.

Biomarkers of Browning in Cold Exposed Siberian Adults.

Cold-exposure promotes energy expenditure by inducing brown adipose tissue (BAT) thermogenesis, which over time, is also sustained by browning, the appearance, or increase, of brown-like cells into white fat depots. Identification of circulating markers reflecting BAT activity and browning is crucial to study this phenomenon and its triggers, also holding possible implications for the therapy of obesity and metabolic diseases. Using RT-qPCR, we evaluated the peripheral blood mononuclear cells (PBMC) expression profile of regulators of BAT activity (CIDEA, PRDM16), white adipocytes browning (HOXC9 and SLC27A1), and fatty acid ß-oxidation (CPT1A) in 150 Siberian healthy miners living at extremely cold temperatures compared to 29 healthy subjects living in thermoneutral conditions. Anthropometric parameters, glucose, and lipid profiles were also assessed. The cold-exposed group showed significantly lower weight, BMI, hip circumference, and PBMC expression of CIDEA, but higher expression of HOXC9 and higher circulating glucose compared to controls. Within the cold-exposed group, BMI, total cholesterol, and the atherogenic coefficient were lower in individuals exposed to low temperatures for a longer time. In conclusion, human PBMC expresses the brown adipocytes marker CIDEA and the browning marker HOXC9, which, varying according to cold-exposure, possibly reflect changes in BAT activation and white fat browning.

The synovial surface of the articular cartilage.

The articular cartilage has been the subject of a huge amount of research carried out with a wide array of different techniques. Most of the existing morphological and ultrastructural data on the this tissue, however, were obtained either by light microscopy or by transmission electron microscopy. Both techniques rely on thin sections and neither allows a direct, face-on visualization of the free cartilage surface (synovial surface), which is the only portion subject to frictional as well as compressive forces. In the present research, high resolution visualization by scanning electron microscopy and by atomic force microscopy revealed that the collagen fibrils of the articular surface are exclusively represented by thin, uniform, parallel fibrils evocative of the heterotypic type IX-type II fibrils reported by other authors, immersed in an abundant matrix of glycoconjugates, in part regularly arranged in phase with the D-period of collagen. Electrophoresis of fluorophore-labeled saccharides confirmed that the superficial and the deeper layers are quite different in their glycoconjugate content as well, the deeper ones containing more sulfated, more acidic small proteoglycans bound to thicker, more heterogenous collagen fibrils. The differences found between the synovial surface and the deeper layers are consistent with the different mechanical stresses they must withstand.

Normal human macula densa morphology and cell turnover: A histological, ultrastructural, and immunohistochemical investigation.

The aim was to analyze the morphology of normal human macula densa (MD), evaluate the cells that may be responsible for its turnover, and collect quantitative data. Of four samples of normal human renal tissue, two were embedded in resin to measure the longitudinal extension and examine the ultrastructure of the MD, the other two were embedded in paraffin to study apoptosis and cell proliferation. The MD is composed of a monolayer tissue about 40 µm long, which includes 35-40 cells arranged in overlapping rows. Ultrastructurally, MD cells show two polarized portions: an apical end, with sensory features, and a basolateral aspect, with paracrine function. MD cells are connected apically by tight junctions, with/without adherens junctions, which form a barrier between the distal tubule lumen and the interstitium. Cells in degeneration, often associated with macrophages, and undifferentiated cells were found in the MD and adjacent distal tubule. A filamentous mat previously described in proximal tubule scattered tubular cells (STCs) was detected in the basal cytoplasm in undifferentiated cells. The tissue was consistently negative for the proliferation marker Ki67 and for the apoptotic markers caspase-3 and caspase-9. This work confirms our earlier morphological findings and provides new data: (a) MD cells display both apical adherens and tight junctions, the latter forming a tubulo-mesangial barrier; (b) the MD is a monolayer made up of about 40 cells arranged in rows; (c) the simultaneous presence of degenerating (8-13%) and undifferentiated (4-13%) cells reminiscent of STCs suggests a non-negligible cell turnover.

Remodeling of collagen constituting interlobular septa of subcutaneous adipose tissue following microwaves application.

In this study, the application of a recently introduced device based on electromagnetic energy transfer by microwaves for fat reduction, permitted to study specifically the modifications of thick fibrous collagen interlobular septa in the subcutaneous adipose tissue, related to the formation of large clusters of adipocytes. The use of Picrosirius red staining associated with circularly polarized microscopy gave evidence of appreciable modifications of the fibrous connective tissue forming septa. Compact fibrotic bundles of collagen I forming interlobular septa appeared reduced or dissolved, in part substituted by the increase of more diffuse and finely reticular collagen III. Remodeling of fibrous collagen, which formed bridles involved in the appearance at the surface of the skin of dimpling/orange peer pattern typical of cellulite, was observed.

A large proportion of mediastinal and perirenal visceral fat of Siberian adult people is formed by UCP1 immunoreactive multilocular and paucilocular adipocytes.

Many deleterious consequences for health of excessive fat accumulation are due to visceral fat. Browning of visceral fat is mainly cold dependent and has been proposed as a possible tool for future therapies of obesity and related disorders. In this paper, we studied the composition of mediastinal and perirenal visceral fat, collected at necropsy, of human adults that lived in Siberia, one of the coldest regions of the earth. Data showed that a consistent part of the mediastinal and perirenal fat (up to about 40%) had the morphology typical of brown adipocytes and that a relevant percentage of them (up to about 30%) also expressed the functional marker uncoupling protein 1 (UCP1). Patients living mainly outdoor had higher percentage of brown-like adipocytes with more intensely UCP1 immunoreactive cells. The presence of numerous UCP1 immunoreactive paucilocular cells, a transitional stage of transdifferentiating adipocytes, supports the idea that visceral fat can be converted to brown adipose tissue in adult humans in physiological conditions. Tyrosine hydroxylase immunoreactive noradrenergic parenchymal nerve fibers were positively correlated to the number of multilocular adipocytes in mediastinal fat, and a similar trend was also observed in the perirenal fat.

New System Delivering Microwaves Energy for Inducing Subcutaneous Fat Reduction: In - Vivo Histological and Ultrastructural Evidence.

BACKGROUND: Recently, it has been developed a new technology for the reduction of subcutaneous adipose tissue through a non-invasive treatment by microwaves. The main objective of the present study is to demonstrate the feasibility of utilising a non-invasive, localised microwaves (MW) device to induce thermal modifications into subcutaneous adipose tissue only by a controlled electromagnetic field that heats up fat preferentially. This device is provided with a special handpiece appropriately cooled, directly contacting the cutaneous surface of the body, which provides a calibrated energy transfer by microwaves. AIM: In this paper, microscopic and ultrastructural modifications of subcutaneous adipose tissue induced by microwaves irradiation are evaluated. METHODS: Our experimental plan was designed for collecting biopsy samples, for each skin region treated with a single irradiation session, 1) before treatment (control), 2) immediately after treatment, 3) after 6 hrs, 4) after 1 month, 5) after 2 months. Bioptic samples from each step were processed for light microscopy and transmission electron microscopy. At the same time, each region where biopsies were collected was subjected to ultrasound examination. Recorded images permitted to evaluate the thickness of different layers as epidermis, dermis, hypodermis, connective fasciae, until to muscle layer, and related modifications induced by treatment. RESULTS: In every biopsy collected at different time-steps, epidermis and superficial dermis appeared not modified compared to control. Differently, already in the short-term biopsies, in the deep dermis and superficial hypodermis, fibrillar connective tissue appeared modified, showing reduction and fragmentation of interlobular collagen septa. The most important adipose tissue modifications were detectable following 1 month from treatment, with a significant reduction of subcutaneous fat, participating both the lysis of many adipocytes and the related phagocytic action of monocytes/macrophages on residuals of compromised structures of adipocytes. In the samples collected two months following treatment, the remnants of adipose tissue appeared normal, and macrophages were completely absent. CONCLUSIONS: Ultrasound, microscopic and ultrastructural evidence are supporting significant effectiveness of the new device treatment in the reduction of subcutaneous fat. In this paper, the possible mechanisms involved in the activation of the monocytes/macrophages system responsible for the removal of adipocytes residuals have also been discussed.

Is there an association between drugs and burning mouth syndrome? A case-control study.

OBJECTIVE: The purpose of this case-control study was to compare the pharmacological anamnesis collected from a group of 150 burning mouth syndrome (BMS) patients with that of a control group of 150 patients matched for age and sex. MATERIALS AND METHODS: The patients' medical histories were reviewed, and data on drug therapy were collected. Drugs were classified on the basis of pharmacological effects; the classes were antihypertensives (i.e., ACE inhibitors/ARBs, calcium antagonists, diuretics and beta-blockers), antiaggregants, anticoagulants, antidiabetics, vitamin D integrators, bisphosphonates, psychotropics (i.e., anxiolytics and antidepressants), gastroprotectors, statins, thyroid hormone substitutes, corticosteroids and immunosuppressants. RESULTS: The BMS patients and the controls were matched for age (mean age: 69 years) and sex (128 females and 22 males). Antihypertensives, especially ACE inhibitors/ARBs (OR = 0.37, CI: 0.22-0.63, p = 0.0002) and beta-blockers (OR = 0.36, CI: 0.19-0.68 p = 002), revealed an inverse association with the presence of BMS, whereas anxiolytics (OR = 3.78, CI: 2.12-6.75 p < 0.0001), but neither antidepressants nor antipsychotics, were significantly associated with BMS. There were no correlations with other drug classes. CONCLUSION: Our study highlighted that ACE inhibitors, ARBs and beta-blockers were in inverse relation to BMS and found that anxiolytics, but neither antidepressants nor antipsychotics, were linked to the presence of the syndrome.

Visualizing the Supramolecular Assembly of Collagen.

Among biological macromolecules, collagen enjoys quite a peculiar status. Making up as much as a third of the protein fraction of the body it is the main responsible for the functional properties of the extracellular matrix, which can be efficiently tuned and tailored by modifying the length, volume fraction, and spatial layout of its collagen content. The supramolecular aggregates of collagen are therefore subject to be investigation by several viewpoints and at different scales, from the finest interactions of individual collagen molecules to the spatial layout of fibril bundles. As a consequence, no treatise can pretend to be exhaustive about the several techniques that can be useful in different moments and/or for different purposes. So, in this chapter, we focus only on some applications of the transmission electron microscope (TEM), of the scanning electron microscope (SEM), and of the atomic force microscope (AFM).

Arterial and microvascular supply of cerebral hemispheres in the nude mouse revealed using corrosion casting and scanning electron microscopy.

Morphological analyses of cerebral vascularization are not only important for the characterization of the anatomical and physiological relationships between vascular and nervous tissue, but also required to understand structural modifications that occur in many pathological conditions affecting the brain. The aim of this study was to generate a three-dimensional vascular map of the cerebral hemispheres in the nude mouse brain, a widely used animal model for studying tumour biology. We used the corrosion casting (CC) technique to isolate blood vessels from 30 nude mouse brains. All casts were analysed using scanning electron microscopy (SEM), which generated quantitative data regarding vessel length and diameter as well as inter-vascular and inter-branching distances. We identified three different topographical regions: (i) the cortical region, characterized by a superficial wide sheet of vessels giving rise to terminal perforant vessels that penetrate the grey matter; (ii) the inner part of the grey matter, in which dense capillary nets form many flake-like structures extending towards the grey-white matter boundary, where perforant vessels finally change direction and form a well-defined vascular sheet; and (iii) the white matter layer, characterized by a more disorganized vascular architecture. In this study, we demonstrate the accuracy of the CC-SEM method in revealing the 3D-topographical organization of the vascular network of the normal nude mouse brain. These baseline data will serve as a reference for future anatomical investigations of pathological alterations, such as tumour infiltrations, using the nude mouse model.