Scanning electron microscopy and X-ray microprobe analysis in detection of acetylcholinesterase in cultured embryonal carcinoma cells.

Abstract

Mouse F9 cells, induced by retinoic acid and dibutyryl cyclic adenosine monophosphate (cAMP) to differentiate into neural-type cells, were incubated for localization of specific acetylcholinesterase (AChE) activity according to the Karnovsky-Roots method where the final enzymatic reaction product is crystalline cupric ferrocyanide and cuprous thiocholine iodide. By scanning electron microscopy (SEM) neural-type cells with long processes were seen. Most of these cells exhibited crystalline precipitates on their surface that in microprobe analysis contained copper, iron, and sulfur. These elements were also detected in some of the neural-type cells that had no visible surface precipitates. Thus, the X-ray analysis also revealed intracellular enzymatic activity. Undifferentiated rounded cells, devoid of AChE activity at the light microscope level, did not show any surface precipitates by SEM and lacked copper, iron, and sulfur emission peaks in the elementary analysis. These results demonstrate that elementary analysis of cytochemical enzymatic reaction products by SEM can be used in identifying cells.