Sébastien Barré

Visualization and stereological characterization of individual rat lung acini by high-resolution X-ray tomographic microscopy

The small trees of gas-exchanging pulmonary airways, which are fed by the most distal purely conducting airways, are called acini and represent the functional gas-exchanging units. The three-dimensional architecture of the acini has a strong influence on ventilation and particle deposition. Due to the difficulty in identifying individual acini on microscopic lung sections, the knowledge about the number of acini and their biological parameters, like volume, surface area, and number of alveoli per acinus, are limited. We developed a method to extract individual acini from lungs imaged by high-resolution synchrotron radiation-based X-ray tomographic microscopy and estimated their volume, surface area, and number of alveoli. Rat acini were isolated by semiautomatically closing the airways at the transition from conducting to gas-exchanging airways. We estimated a mean internal acinar volume of 1.148 mm(3), a mean acinar surface area of 73.9 mm(2), and a mean of 8,470 alveoli/acinus. Assuming that the acini are similarly sized throughout different regions of the lung, we calculated that a rat lung contains 5,470 ± 833 acini. We conclude that our novel approach is well suited for the fast and reliable characterization of a large number of individual acini in healthy, diseased, or transgenic lungs of different species, including humans.

Dose optimization approach to fast X-ray microtomography of the lung alveoli

A framework for linking image quality to radiation dose in order to optimize experimental parameters with respect to dose reduction is presented.

Efficient estimation of the total number of acini in adult rat lung.

Pulmonary airways are subdivided into conducting and gas‐exchanging airways. An acinus is defined as the small tree of gas‐exchanging airways, which is fed by the most distal purely conducting airway. Until now a dissector of five consecutive sections or airway casts were used to count acini. We developed a faster method to estimate the number of acini in young adult rats. Right middle lung lobes were critical point dried or paraffin embedded after heavy metal staining and imaged by X‐ray micro‐CT or synchrotron radiation‐based X‐rays tomographic microscopy. The entrances of the acini were counted in three‐dimensional (3D) stacks of images by scrolling through them and using morphological criteria (airway wall thickness and appearance of alveoli). Segmentation stopper were placed at the acinar entrances for 3D visualizations of the conducting airways. We observed that acinar airways start at various generations and that one transitional bronchiole may serve more than one acinus. A mean of 5612 (±547) acini per lung and a mean airspace volume of 0.907 (±0.108) μL per acinus were estimated. In 60‐day‐old rats neither the number of acini nor the mean acinar volume did correlate with the body weight or the lung volume.

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.

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.

Morphological Aspects of Tumor Angiogenesis

The tumor vasculature is a chaotic mixture of abnormal, hierarchically disorganized vessels that differ from those of normal tissues with respect to organization, structure and function. Firstly, tumor vessel wall structure is abnormal and heterogeneous within the tumor. Besides contractile wall components, the perivascular compartment is often lacking pericytes, what makes the tumor vessels fragile and leaky. Secondly, another group of abnormalities involves distortions in angioarchitecture and vasculature as network. Common features of tumor vessels, irrespective of their origin, size and growth pattern, are absence of hierarchical organization, formation of vessels with irregular contours and their heterogeneous distribution within the tumor.

High‐Resolution Phase‐Contrast Imaging of Submicron Particles in Unstained Lung Tissue

To access the risks and chances of deposition of submicron particles in the gas-exchange area of the lung, a precise three-dimensional (3D)-localization of the sites of deposition is essential--especially because local peaks of deposition are expected in the acinar tree and in individual alveoli. In this study we developed the workflow for such an investigation. We administered 200-nm gold particles to young adult rats by intratracheal instillation. After fixation and paraffin embedding, their lungs were imaged unstained using synchrotron radiation x-ray tomographic microscopy (SRXTM) at the beamline TOMCAT (Swiss Light Source, Villigen, Switzerland) at sample detector distances of 2.5 mm (absorption contrast) and of 52.5 mm (phase contrast). A segmentation based on a global threshold of grey levels was successfully done on absorption-contrast images for the gold and on the phase-contrast images for the tissue. The smallest spots containing gold possessed a size of 1-2 voxels of 370-nm side length. We conclude that a combination of phase and absorption contrast SRXTM imaging is necessary to obtain the correct segmentation of both tissue and gold particles. This method will be used for the 3D localization of deposited particles in the gas-exchange area of the lung.

Cutting-edge microangio-CT: new dimensions in vascular imaging and kidney morphometry

In the last decades, the contrast-enhanced micro-computed tomography (micro-CT) imaging of a whole animal kidney became increasingly important. The visualization was mainly limited to middle-sized vessels. Since modern desktop micro-CT scanners provide the necessary detail resolution, we developed an approach for rapid visualization and consistent assessment of kidney vasculature and glomeruli number. This method is based on μAngiofil, a new polymerizing contrast agent with homogenous X-ray absorption, which provides continuous filling of the complete vasculature and enables correlative imaging approaches. For rapid and reliable kidney morphometry, the microangio-CT (µaCT) data sets from glial cell line-derived neurotrophic factor (GDNF)+/- mice and their wild-type littermates were used. The results were obtained much faster compared with the current gold standard, histology-based stereology, and without processing artifacts. The histology-based morphometry was done afterward on the same kidneys. Both approaches revealed that the GDNF+/- male mice had about 40% fewer glomeruli. Furthermore, our approach allows for the definition of sites of interest for further histological investigation, i.e., correlative morphology. The polymerized μAngiofil stays in perfused vessels and is autofluorescent, which is what greatly facilitates the matching of histological sections with µaCT data. The presented approach is a time-efficient, reliable, qualitative, and quantitative methodology. Besides glomerular morphometry, the µaCT data can be used for qualitative and quantitative analysis of the kidney vasculature and correlative morphology.