Using electron microscopes to look into the lung

Abstract

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.

The micromechanics of lung alveoli: structure and function of surfactant and tissue components

“…Hence, if the volume change within an alveolus results in a real stretching of cells in the inter-alveolar septa or is linked with an unfolding of pleats and shape changes remains unanswered by these investigations. In-depth analyses to answer these questions would require electron microscopic resolution (for review, see Ochs et al 2016) to visualize the basal lamina (and the epithelial cells) which might be stretched so that its surface area increases or simply unfold as ultrastructural investigations from fixed lungs suggest. Until now, however, there is no direct visual evidence whether these mechanisms are really involved during spontaneous or mechanical ventilation in a living subject.…”

“…The mammalian lung is a paradigmatic example for this principle. It is a complex organ whose functional capacity as a gas exchanger is directly determined by its microstructure (for review, see Weibel 1984, 2017; Ochs and Weibel 2015; Ochs et al 2016; Hsia et al 2016). In humans, at the end of a deep inspiration, over 80% of the total volume of the lung is air, and about 10% is blood.…”

The Three-Dimensional Ultrastructure of the Human Alveolar Epithelium Revealed by Focused Ion Beam Electron Microscopy

“…Volume electron microscopic (EM) techniques for 3D modeling of biological structures, including "conventional" techniques like single sectioning transmission electron microscopy (ssTEM) and "new" techniques like SBF SEM and FIB SEM, have been reviewed extensively recently, e.g., [9][10][11][12][13] and some of them have already been used for 3D reconstructions of lung structure: ssTEM [5,7,8,14]; electron tomography [15]; array tomography [16] and SBF SEM [6,11]. Even FIB SEM has been applied [17][18][19], but the study of Købler et al was rather focused on testing an alternative method to diamond knife ultramicrotomy and transmission electron microscopic imaging of nanotubes; the review of Ochs et al gave an outlook on the potential of this technique in lung research; and Hegermann et al demonstrated a method for correlative light and electron microscopy. Kremer et al [11] showed a FIB SEM-based reconstruction of a small part of a human A549 cell.…”

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

“…Serial block-face scanning electron microscopy (SBF-SEM) in combination with digital image analysis offers a method to analyze the ACN of newborn lungs. In contrast to serial sectioning, this technique is based on the repetitive scanning of a block face followed by (automatic) removal of an ultra-thin (approximately 80 nm) section (Peddie and Collinson, 2014; Ochs et al, 2016; Schneider et al, 2019). Scans can run automatically over a few days to weeks and are nearly free of sectioning artifacts.…”