H. Fehrenbach

Studying lung ultrastructure

The study of ultrastructure comprises a wide field of techniques and instrumentations that are used to investigate the organisation of an organ, its tissues, and their cells at the subcellular level all the way down to molecules. The central theme is: How are these components organized in space, i.e. how do they look in three dimensions, and how are they arranged in 3-D with respect to one another?

Ultrastructural pathology of the alveolar type II pneumocytes of human donor lungs

Abstractu2002Alveolar typexa0II pneumocytes (PII) were studied in 12 human donor lungs perfused with modified Euro-Collins solution during single-lung transplantation (SLTx). While one lung was transplanted, the contralateral donor lung (cDL) was fixed at the time of SLTx for examination by electron microscopy, stereology, and microanalysis. Three groups were then formed: group A (nxa0=xa07), cDL without contusions, uneventful early postoperative course; group B (nxa0=xa03), cDL with contusions, uneventful early postoperative course; group C (nxa0=xa02), cDL without contusions, early postoperative respiratory dysfunction. The major findings were that the presence of contusions had no effect on PII ultrastructure and that intracellular surfactant-storing lamellar bodies of cDL in groupxa0C were characterized by a higher volume-to-surface ratio (VsR) and larger area per cell profile than group A. Correlation analysis based on pooled data (groups A and C) showed that ischaemic time had little effect on PII ultrastructure and bore no relationship to postoperative clinical variables. The duration of preoperative donor intubation had a pronounced influence on ultrastructure and postoperative clinical variables. The stereologically estimated amount of intracellular surfactant and mitochondrial VsR were the only ultrastructural parameters that were significantly associated with early postoperative oxygenation. Lamellar bodies were the only ultrastructural components found to have a significant relationship to postoperative intubation time. The ultrastructural integrity of typexa0II pneumocytes of human donor lungs is an important determinant of early respiratory function following clinical lung transplantation.

Improved preservation of phospholipid-rich multilamellar bodies in conventionally embedded mammalian lung tissue--an electron spectroscopic study.

Different conventional methods of tissue processing were studied to determine the extent to which phospholipid‐rich multilamellar bodies of pulmonary alveolar epithelial type II cells of the pig were preserved. Prolonged treatment with half‐saturated aqueous uranyl acetate yielded excellent results on the stabilization of the multilamellar substructure, irrespective of whether glutaraldehyde‐paraformaldehyde or glutaraldehydetannic acid was used as a primary fixative. The lamellar periodicities were observed to be 5·5–6·1 nm.

Electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) of multilamellar bodies and multilamellar body-like structures in tannic acid-treated alveolar septal cells.

We used electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) to compare multilamellar bodies (MLB) of Type II alveolar epithelial cells with MLB-like structures that are present in various alveolar septal cells after fixation with tannic acid. Despite their structural similarity in conventional transmission electron microscopy, the phosphorus signal recorded by both ESI and EELS was considerably higher in multilamellar bodies than in MLB-like structures. This indicates that they are different in chemical composition.

Evaluation of lanthanide tracer methods in the study of mammalian pulmonary parenchyma and cardiac muscle by electron energy-loss spectroscopy

Lanthanum (La) has widely been used as a tracer to study the integrity of plasma membranes. With conventional transmission electron microscopy (cTEM), the absence of electron scattering deposits from the cytoplasm has generally been assumed to reflect an intact cell membrane. However, the application of electron spectroscopic imaging (ESI) and electron energy‐loss spectroscopy (EELS) reveals that electron scattering deposits may be present which do not contain La. However, La could be detected in regions of pulmonary parenchyma and cardiac muscle that were devoid of electron scattering deposits. Therefore, to exclude misinterpretations based on cTEM the application of microanalytical techniques is strongly recommended for the study of the integrity of plasma membranes by means of La tracers. In addition, ESI and EELS are shown to distinguish between different tracers in simultaneous applications of La and terbium (Tb) which were used at the different faces of the pulmonary air‐blood barrier. The analysis of the distribution of both tracers which form electron scattering deposits, indistinguishable by cTEM, may help us to understand the different functional significances of cellular alterations of both cellular borders of the barrier.

Electron spectroscopic study (ESI, EELS) of Nanoplast-embedded mammalian lung.

The potential of Nanoplast melamine resin embedding for the study of mammalian lung parenchyma was examined by means of electron spectroscopic imaging (ESI) and electron energy‐loss spectroscopy (EELS). Samples were either fixed with glutaralde‐hyde‐paraformaldehyde or glutaraldehyde‐tannic acid, or were directly transferred to the embedding medium without prior fixation. Organic dehydrants, as well as fixatives containing heavy metals and stains, were omitted.

Scanning and transmission electron microscopy of human donor lungs: fine structure of the pulmonary parenchyma following preservation and ischemia.

In six cases of clinical single-lung transplantation, the contralateral donor lungs were studied by means of scanning (SEM) and transmission (TEM) electron microscopy. SEM and TEM revealed an overall good to excellent preservation of the pulmonary parenchyma. Since the donor double lung blocks, which were preserved by means of pulmonary arterial perfusion with modified Euro-Collins solution, were separated immediately prior to transplantation, transplanted and contralateral lungs had been subjected to identical influences. Comparing the arterial oxygenation achieved in the recipient 6 h after transplantation with the structural data of the corresponding contralateral donor lung, the degree of epithelial alterations appeared to be most important in determining postoperative lung function. Concerning the assessment of the quality of the alveolar epithelium, gradings performed independently by means of SEM and TEM yielded largely corresponding scores, while a significant shortcoming of SEM was the very limited access to the endothelial surface. Taking into account the relevance of epithelial injury in determining postoperative lung function, SEM can provide clinically relevant data more quickly than TEM. To quantify the fine structural alterations of human donor lungs, however, TEM-based morphometry is required.

Micro analysis of the egg shell of Adela metallica (Poda) (Lepidoptera : Adelidae) by energy-filtering transmission electron microscopy (EFTEM)

Abstract The egg shell of the incurvarioid moth Adela metallica (Lepidoptera : Adelidae) was studied by conventional (cTEM) and energy-filtering transmission electron microscopy (EFTEM). The shell of the laid egg consists of 3 envelopes. The vitelline envelope is 0.1–0.2μm thick and homogeneous, thus exhibiting the non-exoporian character state. The single-layered chorion, which is covered by a fibrogranular mucous layer, is 0.5–0.9μm thick and homogeneous, thus exhibiting the non-ditrysian character state. The chorion is highly electron-lucent. Neither cTEM nor EFTEM revealed any sub-structural details. However, electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS), revealing the elemental composition of the egg shell, indicate that the chorion and vitelline envelope are proteinaceous and hence, similar to the egg shells of other lepidopteran species. The presence of high sulphur signals associated with the vitelline envelope and the thin basal lamella of the chorion indicates that these components may be stabilized via sulphur-bridges.