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

Abstract

SUMMARY 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. A very high level of ultrastructural detail of chromatin, ribosomes, mitochondria and plasma membranes was achieved by ESI from the Nanoplast‐embedded samples. The most prominent gain in ultrastructural detail was achieved when moving from an energy loss just below the L2,3 edge of phosphorus at 132 eV to an energy loss just beyond this edge. This reflects the prominent P L2,3 edge observed by EELS of Nanoplast‐embedded samples in comparison with conventionally processed samples. Thus, taking into account possible sectioning artefacts, excellent heterochromatin images which rely on the phosphorus distribution can be obtained from Nanoplast‐embedded samples by computer‐assisted analysis of electron spectroscopic images. In this respect glutaraldehyde‐paraformaldehyde fixation is preferable to glutaraldehyde‐tannic acid fixation because the presence of silicon, revealed by EELS, in tannic‐acid‐fixed samples may introduce artefacts in phosphorus distribution images obtained by the three‐window method because of the close proximity of the L2,3 edges of silicon and phosphorus.

Immunocytochemical approach for surface layer proteins of freeze-substituted Tannerella forsythensis by energy-filtering transmission electron microscopy

“…Post-fixation by potassium ferrocyanide-reduced OsO 4 has been widely applied to obtain fine membrane structures with a clear contrast for morphological observation by transmission electron microscopy (Doine et al, 1984;Rambourg et al, 1993). However, we found that postfixed Tannerella forsythensis, a bacterium associated with periodontitis, followed by potassium ferrocyanidereduced OsO 4 showed no retention of immunocytochemical reactivity. It was found that 230 and 270-kDa proteins might be the component of the S-layer, based upon the results of the immunoelectron microscopic study of 4% paraformaldehyde-0.05% glutaraldehyde fixation without OsO 4 or freeze-substituted method.…”

“…As previously documented, ESI and EELS analyses under the EF-TEM have been applied to demonstrate isolated ferritin particles (Beckers et al, 1994), calcium elements of plant root caps (Busch et al, 1993), iodinecontaining anti-cancer drug meta-iodobenzyl guanidine (Huxham et al, 1992), P elements in mammalian lung paraenchyma (Fehrenbach et al, 1992) and cyanobacteria (Golecki and Heinrich, 1991), and calcium-ATPase in synaptic terminals (Körtje et al, 1991). These methods are fairy reliable and useful to specifically analyze several elements in biological materials and especially superior in a mapping analysis compared to an X-ray microanalysis.…”

“…Therefore, several investigations have used potassium ferrocyanide-reduced OsO 4 by which an excellent preservation of the ultrastructure and good immunoreactivity of the antigens became possible (Tamaki and Yamashina, 1994;Tamatani et al, 1995). Post-fixation by potassium ferrocyanide-reduced OsO 4 has been widely applied to obtain fine membrane structures with a clear contrast for morphological observation by transmission electron microscopy (Doine et al, 1984;Rambourg et al, 1993). However, we found that postfixed Tannerella forsythensis, a bacterium associated with periodontitis, followed by potassium ferrocyanidereduced OsO 4 showed no retention of immunocytochemical reactivity.…”

Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins fromTannerella forsythensisusing microwave or autoclave heating with citraconic anhydride

“…The immunocytochemical localization of colloidal gold particle-conjugated secondary antibodies corresponded to the nitrogen identified by the maximal electron energy loss at 410 eV (K edge) in the ESI analysis. As reported earlier, ESI and EELS analyses under the EFTEM have been applied to demonstrate isolated ferritin particles (Beckers et al 1994), calcium elements of plant root caps (Busch et al 1993), iodine-containing anti-cancer drug meta-iodobenzyl guanidine (Huxham et al 1992), phosphorus in mammalian lung parenchyma (Fehrenbach et al 1992) and cyanobacteria (Golecki and Heinrich 1991), and calcium-ATPase in synaptic terminals (Kortje et al 1991). These methods are fairly reliable and useful for analyzing several elements in biological materials; they are especially useful for mapping analysis compared to X-ray microanalysis.…”

“…That is, peroxidase-DAB reaction leads to DAB polymerization via nitrogen atoms (Vannier-Santos and Lins 2001). Two different modes of the EFTEM (Golecki and Heinrich 1991, Kortje et al 1991, Fehrenbach et al 1992, Huxham et al 1992, Busch et al 1993, Beckers et al 1994) have been used for electron spectroscopic images (ESI) and electron energy-loss spectroscopy (EELS). Parallel-EELS is an additional tool.…”