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.