Application of scanning electron microscopy to x-ray analysis of frozen-hydrated sections. II. Analysis of standard solutions and artificial electrolyte gradients.

Abstract

New specimen handling and analytic techniques for the application of x- ray microanalysis to studies of cell and organ biology have been recently described (Saubermann et al., 1981, J. Cell Biol. 88:257-267). Based on these techniques, absolute quantitative standardization has been established through x-ray analysis of frozen-hydrated and then dried sections of independently measured standard solutions of elements. These experiments demonstrate that the specific techniques employed have a probable error of less than 10%. Artificial electrolyte gradients established in gelatin were subjected to analysis to determine whether there was elemental displacement under non-membrane- limited conditions at the temperatures employed for sectioning (-30 degrees to -40 degrees C). No significant difference was observed between such gradients in serial sections cut at -30 degrees and -80 degrees C. Similarly, no additional ice-crystal-damage artifact was found in sections cut at -30 degrees C when compared with sections cut at -80 degrees C. Thus, in terms of ice-crystal size, gradient maintenance, and compartmental differentiation, cryosectioning at -30 degrees to -40 degrees C was not associated with redistribution incompatible with 1- to 2-micrometers spatial resolution, and absolute measurements of elemental concentration were practical within regions of this size.

Intracellular Na+:K+ ratios in human cancer cells as revealed by energy dispersive x-ray microanalysis.

“…Furthermore, evidence has been published recently (23) showing that the artificial electrolyte gradients prepared at microscopic dimensions in gelatin blocks are not destroyed by redistribution, even when analyzed in frozen-hydrated sections at relatively high temperatures (-30°C) . Because our material was kept at a temperature < -80'C during freeze-drying, we can safely assume that no significant postmortem redistribution of the monovalents could take place during the preparative procedure.…”

Multifunctional mini‐computer program providing quantitative and digital X‐ray microanalysis of cryosectioned biological tissue for the inexperienced analyst

“…Consequently, the options shown are mainly applicable to the continuum normalization 1. Support film white radiation method (Hall, 1979) as modified by Saubermann (Saubermann et al, 1981). RESULTS At present, nearly all of the analyses being performed at the Microprobe Center are being performed on frozen hydrated sections (0.5 pm thick) or frozen dried sections (0.1 to 0.2 pm thick).…”

“…RESULTS At present, nearly all of the analyses being performed at the Microprobe Center are being performed on frozen hydrated sections (0.5 pm thick) or frozen dried sections (0.1 to 0.2 pm thick). The raw data are usually corrected by means of a modified continuum normalization method (Saubermann et al, 1981). A modular program package suitable for use by the inexperienced user, which uses this analytical approach has been written and tested ( Fig.…”

Personal‐computer‐based system for electron beam X‐ray microanalysis of biological samples

“…Samples are quickly frozen from defined physiological states, cryosectioned, and transferred under vacuum to the cold stage of the electron microscopy (Saubermann et al, 1981a). The use of the personal-computer-based system described here is illustrated by measurements on ganglia from leech (Macrobdella decora), which were prepared in a manner similar to that described by Saubermann & Stockton (1988).…”