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

Abstract

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.

Volume electron microscopy: analyzing the lung

“…(see, Ochs and Weibel 2008 ), the lung is an organ that may notably benefit from these techniques. Some studies have already made use of them successfully (e.g., Schneider et al 2018 , 2019 ; Beike et al 2019 ; Buchacker et al 2019 ; Mühlfeld et al 2020 ) and certainly, there are many more to come. They may significantly enhance our comprehension of the lung’s complex microanatomy, shed more light on known and so far unknown structure–function relationships, both under (physiological) mature, developmental and aging conditions, and patho(physio)logic conditions like lung injury and repair (morphological reconstitution of the blood–gas barrier) (cf.…”

“…Due to this limitation of object thickness, ET is a method best suited for subcellular targets smaller than a few hundred nm, which are completely included in one section. Skilled people can succeed in recording and mounting serial tomograms from physical serial sections, and, thus, in obtaining ET reconstructions of thicker objects, for example complete cells (Höög et al 2007 ; Weber et al 2014 ; Mühlfeld et al 2020 ). For such approaches, however, depending on the resolution required, FIB-SEM is often a more efficient solution.…”

“…A recent study on the human alveolar epithelium demonstrates the benefit of FIB-SEM for the visualization of fine structural details: FIB-SEM could image an almost complete AE2 cell and enable detailed reconstructions of a LB secretion event, the microvilli lawn on the cell surface and tiny AE1 cell processes beneath AE2 cells (Schneider et al 2020 ). Also subcellular details in AE2 cells (plate bodies) could be revealed in 3D using FIB-SEM (Mühlfeld et al 2020 ).…”

Stereology and three-dimensional reconstructions to analyze the pulmonary vasculature

“…With the progression of non-destructive imaging methods and digital segmentation tools, these problems may be overcome. In a recent study from our work group, a new approach was established that is based on the combination of micro-computed X-ray tomography (µCT) and light microscopic stereology (Grothausmann et al 2021 ). In short, the left lung of a postnatal rabbit was embedded in a medium that allows later serial sectioning with reproducibly small section thickness (e.g.…”

Introduction: 3D imaging in lung biology

“…The manuscript provides detailed technical descriptions of the data acquisition process, together with data reconstruction and image stitching. Grothausmann et al (2021) combined imaging of a whole rabbit lung with an X-ray source (μCT), with serial section light microscopy imaging to provide a stereological analysis of pulmonary arterial branches. Extensive digital processing was applied to the 3D sets of images, including painstaking registration of the μCT data with the corresponding light microscopy images.…”