A new method for detecting and localizing cell markers endocytosed by fibroblasts in epoxy resin semi-thin sections using scanning electron microscopy combined with energy dispersive X-ray microanalysis after ion-etching

Abstract

Cell marking is widely used to examine cell development and differentiation in developmental biology. We developed a new method for localizing cell markers in a semi-thin epoxy section with scanning electron microscopy. Cultured fibroblasts ingesting carbon particles were autologously transplanted into a rabbit transparent ear chamber, 6 mm in diameter and 100 microm in depth. Eight days after the transplantation, tissues in the chamber were fixed and embedded in epoxy resin. Semi-thin sections were cut and stained with toluidine blue. Fibroblasts in connective tissues which contained black spots were observed with a light microscope. These sections were subsequently ion-etched with an ion-coater and coated with platinum. The same fibroblasts were then visualized by secondary electron imaging using a scanning electron microscope. A nucleus with nuclear envelope, nuclear pores, a nucleolus and heterochromatin, mitochondria with cristae and rough endoplasmic reticulum were observed in the fibroblasts. The black spots in the fibroblasts were identified as bright bodies with the scanning electron microscope. The bright bodies were found to be a lump of tiny particles less than 100 nm in diameter. In order to analyse such particles with energy dispersive X-ray microanalysis, ion-etched sections were coated with carbon. X-ray energy spectrometry clearly demonstrated that these were carbon particles, which had been endocytosed by the fibroblast. This suggests that scanning electron microscopy combined with energy dispersive X-ray microanalysis is useful for detecting carbon particles in the cytoplasm at an ultrastructural level in semi-thin epoxy sections subsequent to ion etching and that this method may be applicable to other cell markers, such as gold particles to track cells in the field of cell development and cell differentiation.

Influence of surface morphology of the kraft pulp fibers on the growth of the transcrystalline layer of polypropylene

“…The XPS is often considered superior to EDXA when nano scale resolutions are desired, but can be disadvantageous when mean O/C ratios at the micrometer scale are needed. Additionally, the quantitative analysis was easy to access with a scanning electron microscope (SEM), and the technique is common in plant and animal cell analysis 22, 23…”

Application of ion etching to immunoscanning electron microscopy

“…To visualize the secondary electron image in this SEM study, an essential IE process under higher ionizing voltage (350 V) was applied to semithin LR White resin sections in comparison with an epoxy resin IE process (300 V) established recently using the Eiko ion coater (Fujiwara et al, 2000;Shimizu et al, 2001) consequent to the harder nature of LR White relative to the epoxy resin. On the other hand, when very mild, rapid supplementary IE (300 V, 1 min) was performed prior to immunolabeling, artifacts were reduced.…”

“…That is, in instances of fragile or minute cellular organelles, mild IE over an extended period requires lower voltage. Structures of interest such as cell borders or organelles, e.g., secretory granules, are readily detected via LM at low magnification and examined in detail at higher magnification by SEM (Fujiwara et al, 2000). Therefore, the IE-immunoSEM technique is suitable for threedimensional reconstruction (Shimizu et al, 2001) of immunostained organelles and for immunoscreening of pathological specimens.…”

“…The IE-SEM method has been examined employing epoxy resin semithin sections (Fujiwara et al, 2000;Shimizu et al, 2001); the findings revealed that an identical section is observable at both LM and SEM levels. A TEM approach involving low magnification was documented by Tandler and Slabe (2002).…”

Immunohistochemical and Immunocytochemical Localization of Amylase in Rat Parotid Glands and von Ebner's Glands by Ion Etching-Immunoscanning Electron Microscopy

“…This is an optimal technique for detecting distributions over a wide area and localization in inner cellular organelles. The structures of interesting organelles, such as SG, are readily detected by light microscopy (LM) at low magnification and examined in detail at high magnification by SEM using the same section [ 5 ].…”