Origin of artifactual quantitation of electrolytes in microprobe analysis of frozen sections of erythrocytes.

Frozen sections of erythrocytes have been used to validate microprobe X-ray analysis of diffusible elements in biological samples. At this meeting last year we reported that intracellular Na concentrations measured by microprobe were much higher than those measured by bulk chemical methods. It was suggested that this might be due to the movement of extracellular material over the cells by microtomy. We now present evidence that such results are better explained by electron scattering within the sample during analysis. This evidence includes observations that the excess measured Na varies directly with section thickness and inversely with accelerating voltage. At 80 kV accelerating voltage there is excellent agreement between microprobe and chemical analysis. It may be concluded that specimen preparation techniques such as used in our laboratory are satisfactory for reliably localizing diffusible elements in small volumes, but that instrumental factors can seriously affect their quantitation.