Ex vivo microangioCT: Advances in microvascular imaging

Therapeutic modulation of angiogenesis is believed to be a prospective powerful treatment strategy to modulate the microcirculation and therefore help millions of patients with cardiovascular and cancer diseases. The often-frustrating results from late-stage clinical studies indicate an urgent need for improved assessment of the pro- and anti-angiogenic compounds in preclinical stage of investigation. For such a proper assessment, detailed vascular visualization and adequate quantification are essential. Nowadays, there are few imaging modalities available, but none of them provides non-destructive 3D-visualization of the vasculature down to the capillary level. In many instances, the approaches cannot be combined with the subsequent histological or ultrastructural analysis. In this review, we address the latest developments in the microvascular imaging, namely, the microangioCT approach with a polymer-based contrast agent (μAngiofil). This approach allows time-efficient non-destructive 3D-imaging of the organ and its vasculature including the finest capillaries. Besides the superior visualization, the obtained detailed 3D information on the organ vasculature enables its 3D-skeletonization and further quantitative analysis. Probably the only significant limitation of the described approach is that it can be used only ex vivo, i.e., no longitudinal studies. In spite of this drawback, microangioCT with μAngiofil is a relatively simple and straightforward tool with a broad application range for studying physiological and pathological alterations in the microvasculature of any organ. It provides microvascular imaging at unprecedented level and enables correlative microscopy

Micro-CT imaging of Thiel-embalmed and iodine-stained human temporal bone for 3D modeling

Introduction This pilot study explores whether a human Thiel-embalmed temporal bone is suitable for generating an accurate and complete data set with micro-computed tomography (micro-CT) and whether solid iodine-staining improves visualization and facilitates segmentation of middle ear structures. Methods A temporal bone was used to verify the accuracy of the imaging by first digitally measuring the stapes on the tomography images and then physically under the microscope after removal from the temporal bone. All measurements were compared with literature values. The contralateral temporal bone was used to evaluate segmentation and three-dimensional (3D) modeling after iodine staining and micro-CT scanning. Results The digital and physical stapes measurements differed by 0.01–0.17 mm or 1–19%, respectively, but correlated well with the literature values. Soft tissue structures were visible in the unstained scan. However, iodine staining increased the contrast-to-noise ratio by a factor of 3.7 on average. The 3D model depicts all ossicles and soft tissue structures in detail, including the chorda tympani, which was not visible in the unstained scan. Conclusions Micro-CT imaging of a Thiel-embalmed temporal bone accurately represented the entire anatomy. Iodine staining considerably increased the contrast of soft tissues, simplified segmentation and enabled detailed 3D modeling of the middle ear

Fossil Carychiidae (Eupulmonata, Ellobioidea) from the Lower Pleistocene Nashua Formation of Florida, with the description of a new species.

Recent fossil shell mining for a new rail line in the Orlando area of Orange County, Florida has uncovered two species of the ellobioid genus Carychium O. F. Müller, 1773 in a bed of freshwater marl from the Lower Pleistocene Nashua Formation. To taxonomically interpret these finds, the well-preserved shells were imaged via high-resolution X-ray tomography (micro-CT) to view significant internal diagnostic characters such as the columellar configuration and the degree of lamellar sinuosity and their relationship in context to the entire shell. The image data are compared to that of type material and extant and fossil Carychium species inhabiting the SE USA, Mexico, Central America, and Jamaica. Based on these results, the species Carychiumfloridanum G. H. Clapp, 1918 and Carychiumnashuaensesp. nov. are identified from fossil shells dating from the Early Pleistocene. This work documents the first fossil members of C.floridanum and the first fossil Carychium from the SE USA

Synchrotron microtomography reveals the fine three-dimensional porosity of composite polysaccharide aerogels

This study investigates the impact of ice-templating conditions on the morphological features of composite polysaccharide aerogels in relation to their mechanical behavior and aims to get a better insight into the parameters governing these properties. We have prepared polysaccharide aerogels of guar galactomannan (GM) and tamarind seed xyloglucan (XG) by enzymatic oxidation with galactose oxidase (GaO) to form hydrogels, followed by conventional and unidirectional ice-templating (freezing) methods and lyophilization to form aerogels. Composite polysaccharide aerogels were prepared by incorporating nanofibrillated cellulose (NFC) into polysaccharide solutions prior to enzymatic oxidation and gel formation; such a cross linking technique enabled the homogeneous distribution of the NFC reinforcement into the gel matrix. We conducted phase-enhanced synchrotron X-ray microtomography (XMT) scans and visualized the internal microstructure of the aerogels in three-dimensional (3D) space. Volume-weighted pore-size and pore-wall thickness distributions were quantitatively measured and correlated to the aerogels’ mechanical properties regarding ice-templating conditions. Pore-size distribution and orientation depended on the ice-templating methods and the NFC reinforcement that significantly determined the mechanical and shape-recovery behavior of the aerogels. The results obtained will guide the design of the microporous structure of polysaccharide aerogels with optimal morphology and mechanical behavior for life-sciences applications.Peer reviewe

Correlative Imaging of the Murine Hind Limb Vasculature and Muscle Tissue by MicroCT and Light Microscopy

A detailed vascular visualization and adequate quantification is essential for the proper assessment of novel angiomodulating strategies. Here, we introduce an ex vivo micro-computed tomography (microCT)-based imaging approach for the 3D visualization of the entire vasculature down to the capillary level and rapid estimation of the vascular volume and vessel size distribution. After perfusion with μAngiofil®, a novel polymerizing contrast agent, low- and high-resolution scans (voxel side length: 2.58-0.66 μm) of the entire vasculature were acquired. Based on the microCT data, sites of interest were defined and samples further processed for correlative morphology. The solidified, autofluorescent μAngiofil® remained in the vasculature and allowed co-registering of the histological sections with the corresponding microCT-stack. The perfusion efficiency of μAngiofil® was validated based on lectin-stained histological sections: 98 ± 0.5% of the blood vessels were μAngiofil®-positive, whereas 93 ± 2.6% were lectin-positive. By applying this approach we analyzed the angiogenesis induced by the cell-based delivery of a controlled VEGF dose. Vascular density increased by 426% mainly through the augmentation of medium-sized vessels (20-40 μm). The introduced correlative and quantitative imaging approach is highly reproducible and allows a detailed 3D characterization of the vasculature and muscle tissue. Combined with histology, a broad range of complementary structural information can be obtained

Percutaneous aortic valve replacement: valvuloplasty studies in vitro

Objective: Valvuloplasty of the aortic valve is currently used in selected patients for severe calcified aortic valve disease, but clinical effectiveness is low and complication rate remains high. In this study, the total particle load after valvuloplasty and the embolization risk of calcific debris into the coronary arteries was analyzed in an in vitro model. Methods: Three highly calcified human aortic leaflets have been sutured into a porcine annulus (N=9). Both coronary arteries were separated and each was anastomized to a silicon line, which was drained off into a measuring beaker. Then valvuloplasty was performed (Thyshak II, 20mm, 1.5atm). After removal of the balloon, 100ml of sodium chloride solution irrigated the ascending aorta. After passing through the separated coronary arteries, the solution was filtered (filter size 0.45μm), dried, and the total amount of particles was analyzed microscopically. Results: Nine experiments were analyzed. After valvuloplasty, all hearts showed a median of 18 particles larger than 1mm in the coronary arteries (range 0-307). The amount of particles smaller than 1mm was 6574 (median, range 2207-14200). In five cases, coronary arteries were completely occluded by bulky particles. Conclusion: This study demonstrated a large amount of calcific particles after valvuloplasty with a consequently high risk for coronary embolic events in case of highly calcified aortic valves. In times of valvuloplasty rediscovering as part of transcatheter valve implantation, the risk of embolization should be taken into consideration and filtering techniques have to be develope

Internal morphology of 101 mandibular canines of a Swiss-German population by means of micro-CT: An ex vivo study

The aim of this study was to investigate the root canal system morphology by means of a root canal configuration (RCC) classification described with a four-digit system, the physiological foramen geometry and accessory canal frequency and morphology, of 101 mandibular canines (MaCa) of a Swiss-German population by means of micro-computed tomography. Micro-CT examination of the MaCa was performed and the obtained images analyzed with a 3D imaging software. In single-rooted MaCas, the most frequently observed RCCs were 1-1-1/1 (74.5%) and 1-1-1/2 (14.3%). Seven other RCCs were less frequently observed with a frequency from 4.1 to 1.0%. One physiological foramen was observed in 80.6% of the MaCas, two in 16.3%, three in 1.0% and four in 2.0%. Accessory and connecting canals were apparent only in the middle and apical root thirds. Two-rooted MaCas occurred less frequently (n = 3). When one physiological foramen was present, the mean size of the narrow and wide diameters were 0.28 mm (±0.07) and 0.40 mm (±0.11), while the distance between physiological and anatomical foramen was 0.45 mm (±0.17). MaCas are predominantly single-rooted teeth with a 1-1-1/1 or 1-1-1/2 RCC. Most MaCas had one physiological foramen with an oval shape

The total number of acini remains constant throughout postnatal rat lung development.

The pulmonary airways are subdivided into conducting and gas-exchanging airways. The small tree of gas-exchanging airways which is fed by the most distal conducting airway represents an acinus. Very little is known about the development of the number of acini. The goal of this study was to estimate their number throughout rat postnatal development. Right middle rat lung lobes were obtained at postnatal day 4-60, stained with heavy metals, paraffin embedded, and scanned by synchrotron radiation-based X-ray tomographic microscopy or imaged with micro computed tomography after critical point drying. The acini were counted by detection of the transitional bronchioles [bronchioalveolar duct junction (BADJ)] by using morphological criteria (thickness of the walls of airways and appearance of alveoli) during examination of the resulting three-dimensional (3D) image stacks. Between postnatal days 4-60, the number of acini per lung remained constant (5,840 ± 547 acini), but their volume increased significantly. We concluded that the acini are formed before the end of the saccular stage (before postnatal day 4) and that the developmental increase of the lung volume is achieved by an increase of the acinar volume and not by an increase of their number. Furthermore, our results propose that the bronchioalveolar stem cells, which are residing in the BADJ, are as constant in their location as the BADJ itself

Radiation dose optimized lateral expansion of the field of view in synchrotron radiation X-ray tomographic microscopy

Increasing the lateral field of view of tomography-based imaging methods greatly increases the acquisition time. This article presents scanning protocols to obtain high-resolution tomographic scans with large lateral field of view at greatly decreased acquisition time and thus reduced radiation dose while resulting in high-quality three-dimensional tomographic datasets

Registration of serial sections: An evaluation method based on distortions of the ground truths

Registration of histological serial sections is a challenging task. Serial sections exhibit distortions and damage from sectioning. Missing information on how the tissue looked before cutting makes a realistic validation of 2D registrations extremely difficult. This work proposes methods for ground-truth-based evaluation of registrations. Firstly, we present a methodology to generate test data for registrations. We distort an innately registered image stack in the manner similar to the cutting distortion of serial sections. Test cases are generated from existing 3D data sets, thus the ground truth is known. Secondly, our test case generation premises evaluation of the registrations with known ground truths. Our methodology for such an evaluation technique distinguishes this work from other approaches. Both under- and over-registration become evident in our evaluations. We also survey existing validation efforts. We present a full-series evaluation across six different registration methods applied to our distorted 3D data sets of animal lungs. Our distorted and ground truth data sets are made publicly available.Comment: Supplemental data available under https://zenodo.org/record/428244