Analysis of the alveolar shape in 3-D.

Mechanical forces affect the alveolar shape, depending on location and tissue composition, and vary during the respiratory cycle. This study performs alveolar morphomics in different lobes of human lungs using models generated from three-dimensional (3-D) micro-computed tomography (microCT) images. Cylindrical tissue samples (1.6 cm × 2 cm) were extracted from two nontransplantable donor lungs (one ex-smoker and one smoker, 3 samples per subject) that were air-inflated and frozen solid in liquid nitrogen vapor. Samples were scanned with microCT (11 µm/voxel). Within representative cubic regions of interest (5.5 mm edge length), alveoli were segmented to produce corresponding 3-D models from which quantitative data were obtained. The surface of segmented alveoli (n_alv_total = 23,587) was divided into individual planar surfaces (facets) and angles between facet normals were calculated. Moreover, the number of neighboring alveoli was estimated for every alveolus. In this study, we examined intraindividual differences in alveolar morphology, which were reproducible in the lungs of two subjects. The main aspects are higher mean alveolar volumes (v_alv: 6.64 × 106 and 6.63 × 106 µm3 vs. 5.78 × 106 and 6.29 × 106 µm3) and surface sizes (s_alv: 0.19 and 0.18 mm2 vs. 0.17 mm2 in both lower lobes) in both upper lung lobes compared with the lower lobes. An increasing number of facets (f_alv) from top to bottom (12 and 14 in the upper lobes; 14 and 15 in the lower lobes), as well as a decreasing number of alveolar neighbors (nei_alv: 9 and 8 in the upper lobes; 8 and 7 in the lower lobes) from the upper lobes to the lower lobes were observed. We could observe an increasing ratio of alveolar entrance size to the surface size of the alveoli from top to bottom (S_ratio_alv: 0.71 and 0.64 in the upper lobes, 0.73 and 0.70 in the lower lobes). The angles between facet normals (ang_alv) were larger in the upper lobes (67.72° and 62.44°) of both lungs than in the lower lobes (66.19° and 61.30°). By using this new approach of analyzing alveolar 3-D data, which enables the estimation of facet, neighbor, and shape characteristics, we aimed to establish the baseline measures for in-depth studies of mechanical conditions and morphology.

Capillary Changes Precede Disordered Alveolarization in a Mouse Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD), the most common sequela of preterm birth, is a severe disorder of the lung that is often associated with long-lasting morbidity. A hallmark of BPD is the disruption of alveolarization, whose pathogenesis is incompletely understood. Here, we tested the vascular hypothesis that disordered vascular development precedes the decreased alveolarization associated with BPD. Neonatal mouse pups were exposed to 7, 14, or 21 days of normoxia (21% O2) or hyperoxia (85% O2) with n = 8-11 for each group. The right lungs were fixed by vascular perfusion and investigated by design-based stereology or three-dimensional reconstruction of data sets obtained by serial block-face scanning EM. The alveolar capillary network of hyperoxia-exposed mice was characterized by rarefaction, partially altered geometry, and widening of capillary segments as shown by three-dimensional reconstruction. Stereology revealed that the development of alveolar epithelium and capillary endothelium was decreased in hyperoxia-exposed mice; however, the time course of these effects was different. That the surface area of the alveolar epithelium was smaller in hyperoxia-exposed mice first became evident at Day 14. In contrast, the surface area of the endothelium was reduced in hyperoxia-exposed mouse pups at Day 7. The thickness of the air-blood barrier decreased during postnatal development in normoxic mice, whereas it increased in hyperoxic mice. The endothelium and the septal connective tissue made appreciable contributions to the thickened septa. In conclusion, the present study provides clear support for the idea that the stunted alveolarization follows the disordered microvascular development, thus supporting the vascular hypothesis of BPD.

Combination of µCT and light microscopy for generation-specific stereological analysis of pulmonary arterial branches: a proof-of-concept study.

Various lung diseases, including pulmonary hypertension, chronic obstructive pulmonary disease or bronchopulmonary dysplasia, are associated with structural and architectural alterations of the pulmonary vasculature. The light microscopic (LM) analysis of the blood vessels is limited by the fact that it is impossible to identify which generation of the arterial tree an arterial profile within a LM microscopic section belongs to. Therefore, we established a workflow that allows for the generation-specific quantitative (stereological) analysis of pulmonary blood vessels. A whole left rabbit lung was fixed by vascular perfusion, embedded in glycol methacrylate and imaged by micro-computed tomography (µCT). The lung was then exhaustively sectioned and 20 consecutive sections were collected every 100 µm to obtain a systematic uniform random sample of the whole lung. The digital processing involved segmentation of the arterial tree, generation analysis, registration of LM sections with the µCT data as well as registration of the segmentation and the LM images. The present study demonstrates that it is feasible to identify arterial profiles according to their generation based on a generation-specific color code. Stereological analysis for the first three arterial generations of the monopodial branching of the vasculature included volume fraction, total volume, lumen-to-wall ratio and wall thickness for each arterial generation. In conclusion, the correlative image analysis of µCT and LM-based datasets is an innovative method to assess the pulmonary vasculature quantitatively.

Assessment of the Alveolar Capillary Network in the Postnatal Mouse Lung in 3D Using Serial Block-Face Scanning Electron Microscopy.

The alveolar capillary network (ACN) has a large surface area that provides the basis for an optimized gas exchange in the lung. It needs to adapt to morphological changes during early lung development and alveolarization. Structural alterations of the pulmonary vasculature can lead to pathological functional conditions such as in bronchopulmonary dysplasia and various other lung diseases. To understand the development of the ACN and its impact on the pathogenesis of lung diseases, methods are needed that enable comparative analyses of the complex three-dimensional structure of the ACN at different developmental stages and under pathological conditions. In this study a newborn mouse lung was imaged with serial block-face scanning electron microscopy (SBF-SEM) to investigate the ACN and its surrounding structures before the alveolarization process begins. Most parts but not all of the examined ACN contain two layers of capillaries, which were repeatedly connected with each other. A path from an arteriole to a venule was extracted and straightened to allow cross-sectional visualization of the data along the path within a plane. This allows a qualitative characterization of the structures that erythrocytes pass on their way through the ACN. One way to define regions of the ACN supplied by specific arterioles is presented and used for analyses. Pillars, possibly intussusceptive, were found in the vasculature but no specific pattern was observed in regard to parts of the saccular septa. This study provides 3D information with a resolution of about 150 nm on the microscopic structure of a newborn mouse lung and outlines some of the potentials and challenges of SBF-SEM for 3D analyses of the ACN.

Recent developments in 3-D reconstruction and stereology to study the pulmonary vasculature.

Alterations of the pulmonary vasculature are an important feature of human lung diseases such as chronic obstructive pulmonary disease, pulmonary hypertension, and bronchopulmonary dysplasia. Experimental studies to investigate the pathogenesis or a therapeutic intervention in animal models of these diseases often require robust, meaningful, and efficient morphometric data that allow for appropriate statistical testing. The gold standard for obtaining such data is design-based stereology. However, certain morphological characteristics of the pulmonary vasculature make the implementation of stereological methods challenging. For example, the alveolar capillary network functions according to the sheet flow principle, thus making unbiased length estimations impossible and requiring other strategies to obtain mechanistic morphometric data. Another example is the location of pathological changes along the branches of the vascular tree. For developmental defects like in bronchopulmonary dysplasia or for pulmonary hypertension, it is important to know whether certain segments of the vascular tree are preferentially altered. This cannot be overcome by traditional stereological methods but requires the combination of a three-dimensional data set and stereology. The present review aims at highlighting the great potential while discussing the major challenges (such as time consumption and data volume) of this combined approach. We hope to raise interest in the potential of this approach and thus stimulate solutions to overcome the existing challenges.

Digital 3D reconstructions using histological serial sections of lung tissue including the alveolar capillary network.

Grothausmann R, Knudsen L, Ochs M, Mühlfeld C. Digital 3D reconstructions using histological serial sections of lung tissue including the alveolar capillary network. Am J Physiol Lung Cell Mol Physiol 312: L243-L257, 2017. First published December 2, 2016; doi:10.1152/ajplung.00326.2016-The alveolar capillary network (ACN) provides an enormously large surface area that is necessary for pulmonary gas exchange. Changes of the ACN during normal or pathological development or in pulmonary diseases are of great functional impact and warrant further analysis. Due to the complexity of the three-dimensional (3D) architecture of the ACN, 2D approaches are limited in providing a comprehensive impression of the characteristics of the normal ACN or the nature of its alterations. Stereological methods offer a quantitative way to assess the ACN in 3D in terms of capillary volume, surface area, or number but lack a 3D visualization to interpret the data. Hence, the necessity to visualize the ACN in 3D and to correlate this with data from the same set of data arises. Such an approach requires a large sample volume combined with a high resolution. Here, we present a technically simple and cost-efficient approach to create 3D representations of lung tissue ranging from bronchioles over alveolar ducts and alveoli up to the ACN from more than 1 mm sample extent to a resolution of less than 1 µm. The method is based on automated image acquisition of serially sectioned epoxy resin-embedded lung tissue fixed by vascular perfusion and subsequent automated digital reconstruction and analysis of the 3D data. This efficient method may help to better understand mechanisms of vascular development and pathology of the lung.

Using electron microscopes to look into the lung.

In the nineteenth century, there was a dispute about the existence of a lung alveolar epithelium which remained unsolved until the invention of electron microscopy (EM) and its application to the lung. From the early 1960s, Ewald Weibel became the master of lung EM. He showed that the alveolar epithelium is covered with a lining layer containing surfactant. Weibel also explained the phenomenon of "non-nucleated plates" observed already in 1881 by Albert Kölliker. Weibel's most significant contribution was to the development of stereological methods. Therefore, quantitative characterization of lung structure revealing structure-function relationships became possible. Today, the spectrum of EM methods to study the fine structure of the lung has been extended significantly. Cryo-preparation techniques are available which are necessary for immunogold labeling of molecules. Energy-filtering techniques can be used for the detection of elements. There have also been major improvements in stereology, thus providing a very versatile toolbox for quantitative lung phenotype analyses. A new dimension was added by 3D EM techniques. Depending on the desired sample size and resolution, the spectrum ranges from array tomography via serial block face scanning EM and focused ion beam scanning EM to electron tomography. These 3D datasets provide new insights into lung ultrastructure. Biomedical EM is an ever-developing field. Its high resolution remains unparalleled. Moreover, EM has the unique advantage of providing an "open view" into cells and tissues within their full architectural context. Therefore, EM will remain an indispensable tool for a better understanding of the lung's functional design.

A combined method for correlative 3D imaging of biological samples from macro to nano scale.

Correlative analysis requires examination of a specimen from macro to nano scale as well as applicability of analytical methods ranging from morphological to molecular. Accomplishing this with one and the same sample is laborious at best, due to deformation and biodegradation during measurements or intermediary preparation steps. Furthermore, data alignment using differing imaging techniques turns out to be a complex task, which considerably complicates the interconnection of results. We present correlative imaging of the accessory rat lung lobe by combining a modified Scanning Laser Optical Tomography (SLOT) setup with a specially developed sample preparation method (CRISTAL). CRISTAL is a resin-based embedding method that optically clears the specimen while allowing sectioning and preventing degradation. We applied and correlated SLOT with Multi Photon Microscopy, histological and immunofluorescence analysis as well as Transmission Electron Microscopy, all in the same sample. Thus, combining CRISTAL with SLOT enables the correlative utilization of a vast variety of imaging techniques.

Lysophosphatidic Acid Signaling through the Lysophosphatidic Acid-1 Receptor Is Required for Alveolarization.

Lysophosphatidic acid (LPA) signaling through one of its receptors, LPA1, contributes to both the development and the pathological remodeling after injury of many organs. Because we found previously that LPA-LPA1 signaling contributes to pulmonary fibrosis, here we investigated whether this pathway is also involved in lung development. Quantitative assessment of lung architecture of LPA1-deficient knock-out (KO) and wild-type (WT) mice at 3, 12, and 24 weeks of age using design-based stereology suggested the presence of an alveolarization defect in LPA1 KO mice at 3 weeks, which persisted as alveolar numbers increased in WT mice into adulthood. Across the ages examined, the lungs of LPA1 KO mice exhibited decreased alveolar numbers, septal tissue volumes, and surface areas, and increased volumes of the distal airspaces. Elastic fibers, critical to the development of alveolar septa, appeared less organized and condensed and more discontinuous in KO alveoli starting at P4. Tropoelastin messenger RNA expression was decreased in KO lungs, whereas expression of matrix metalloproteinases degrading elastic fibers was either decreased or unchanged. These results are consistent with the abnormal lung phenotype of LPA1 KO mice, being attributable to reduced alveolar septal formation during development, rather than to increased septal destruction as occurs in the emphysema of chronic obstructive pulmonary disease. Peripheral septal fibroblasts and myofibroblasts, which direct septation in late alveolarization, demonstrated reduced production of tropoelastin and matrix metalloproteinases, and diminished LPA-induced migration, when isolated from LPA1 KO mice. Taken together, our data suggest that LPA-LPA1 signaling is critically required for septation during alveolarization.

Estimation of the number of alveolar capillaries by the Euler number (Euler-Poincaré characteristic).

The lung parenchyma provides a maximal surface area of blood-containing capillaries that are in close contact with a large surface area of the air-containing alveoli. Volume and surface area of capillaries are the classic stereological parameters to characterize the alveolar capillary network (ACN) and have provided essential structure-function information of the lung. When loss (rarefaction) or gain (angiogenesis) of capillaries occurs, these parameters may not be sufficient to provide mechanistic insight. Therefore, it would be desirable to estimate the number of capillaries, as it contains more distinct and mechanistically oriented information. Here, we present a new stereological method to estimate the number of capillary loops in the ACN. One advantage of this method is that it is independent of the shape, size, or distribution of the capillaries. We used consecutive, 1 µm-thick sections from epoxy resin-embedded material as a physical disector. The Euler-Poincaré characteristic of capillary networks can be estimated by counting the easily recognizable topological constellations of "islands," "bridges," and "holes." The total number of capillary loops in the ACN can then be calculated from the Euler-Poincaré characteristic. With the use of the established estimator of alveolar number, it is possible to obtain the mean number of capillary loops per alveolus. In conclusion, estimation of alveolar capillaries by design-based stereology is an efficient and unbiased method to characterize the ACN and may be particularly useful for studies on emphysema, pulmonary hypertension, or lung development.

Missing wedge computed tomography by iterative algorithm DIRECTT.

A strategy to mitigate typical reconstruction artefacts in missing wedge computed tomography is presented. These artefacts appear as elongations of reconstructed details along the mean direction (i.e. the symmetry centre of the projections). Although absent in standard computed tomography applications, they are most prominent in advanced electron tomography and also in special topics of X-ray and neutron tomography under restricted geometric boundary conditions. We investigate the performance of the DIRECTT (Direct Iterative Reconstruction of Computed Tomography Trajectories) algorithm to reduce the directional artefacts in standard procedures. In order to be sensitive to the anisotropic nature of missing wedge artefacts, we investigate isotropic substructures of metal foam as well as circular disc models. Comparison is drawn to filtered backprojection and algebraic techniques. Reference is made to reconstructions of complete data sets. For the purpose of assessing the reconstruction quality, Fourier transforms are employed to visualize the missing wedge directly. Deficient reconstructions of disc models are evaluated by a length-weighted kernel density estimation, which yields the probabilities of boundary orientations. The DIRECTT results are assessed at different signal-to-noise ratios by means of local and integral evaluation parameters.

Correlating 3D morphology with molecular pathology: fibrotic remodelling in human lung biopsies.

Assessing alterations of the parenchymal architecture is essential in understanding fibrosing interstitial lung diseases. Here, we present a novel method to visualise fibrotic remodelling in human lungs and correlate morphological three-dimensional (3D) data with gene and protein expression in the very same sample. The key to our approach is a novel embedding resin that clears samples to full optical transparency and simultaneously allows 3D laser tomography and preparation of sections for histology, immunohistochemistry and RNA isolation. Correlating 3D laser tomography with molecular diagnostic techniques enables new insights into lung diseases. This approach has great potential to become an essential tool in pulmonary research.

Method for 3D airway topology extraction.

In lungs the number of conducting airway generations as well as bifurcation patterns varies across species and shows specific characteristics relating to illnesses or gene variations. A method to characterize the topology of the mouse airway tree using scanning laser optical tomography (SLOT) tomograms is presented in this paper. It is used to test discrimination between two types of mice based on detected differences in their conducting airway pattern. Based on segmentations of the airways in these tomograms, the main spanning tree of the volume skeleton is computed. The resulting graph structure is used to distinguish between wild type and surfactant protein (SP-D) deficient knock-out mice.

Automated quantitative 3D analysis of faceting of particles in tomographic datasets.

Characterization of facets of particles is a common problem. In this paper an algorithm is presented which allows automated quantitative 3D analysis of facets of many particles within tomographic datasets. The algorithm is based on the analysis of probability distributions of the orientations of triangle normals of mesh representations. The result consists of lists containing number of detected facets, their size, global orientation and the interplanar angles between facets for each analyzed particle. Characterization of each particle according to any of these facet properties is then possible, e.g. statistics about different crystal shapes or removal of particles that do not show significant faceting. Analyses of a 3D dataset obtained by focused ion beam (FIB) tomography of a sample containing spinel particles are presented.

Quantitative structural assessment of heterogeneous catalysts by electron tomography.

We present transmission electron microscope (TEM) tomography investigations of ruthenium-based fuel cell catalyst materials as employed in direct methanol fuel cells (DMFC). The digital three-dimensional representation of the samples not only enables detailed studies on number, size, and shape but also on the local orientation of the ruthenium particles to their support and their freely accessible surface area. The shape analysis shows the ruthenium particles deviate significantly from spherical symmetry which increases their surface to volume ratio. The morphological studies help to understand the structure formation mechanisms during the fabrication as well as the high effectiveness of these catalysts in the oxygen reduction reaction at the cathode side of fuel cells.