Quantification for the X‐ray microanalysis of cryosections

Abstract

SUMMARY Some problems of the quantitative analysis of diffusible elements in cryosections are reviewed. The two prevalent methods for obtaining concentrations from X‐ray data, one based on characteristic radiation alone and the other on continuum‐normalization, are recapitulated. Both methods seem suitable at cellular level while the latter seems preferable at finer spatial resolution. Recourse to both methods together is desirable in the analysis of frozen‐hydrated sections especially when there is no peripheral standard. Selective local contamination is a particular hazard in the analysis of chlorine. In the case of sodium, physical parameters set restrictive limits to the minimum concentration measurable by ‘energy‐dispersive’ X‐ray spectrometry (about 20 mm kg−1) and to the spatial resolution attainable by diffractive X‐ray spectrometry (∼0·2 μm). One obvious danger to meaningful quantitative analysis is inadvertent redistribution of diffusible elements during the moments preceding the freeze‐quenching of a tiny piece of tissue. Data are presented to show that concentration changes due to simple evaporation are a real hazard prior to the quenching of sub‐millimetre size samples.

Tutorial Review: X-ray Mapping in Electron-Beam Instruments

“…Results have been published by several groups~Somlyo, 1984; Fiori et al, 1988;Ingram et al, 1988;Wong et al, 1989;Schultz et al, 1999!, who have usually used the Hall method Hall, 1979;Hall & Gupta, 1982! for converting X-ray intensities to compositions.…”

Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

“…We measured sodium (Na), magnesium (Mg), phosphorus (P), sulfur (S), chloride (Cl), potassium (K) and calcium (Ca) concentrations in the cytoplasm and secretory granules of non-CF and CF cells Xray spectra were acquired for 100 s at 100 keV by scanning the electron probe over a 20 mm 2 in the cytoplasm and nucleus compartment or 0.45 mm 2 surface in the secretory granules. The elemental composition of dehydrated sections was determined with a software based on the Hall continuum method [23] and expressed in mmol per kilogram of dry mass. The water mass content was determined by measuring the quantitative dark field intensity in STEM [20,24].…”

X-ray mapping of freeze-dried cryosections from biological cells

“…Details of preparation and X-ray microanalysis were described previously [3]. Quantitative X-ray microanalytical data in terms of element content related to the dry weight "cd" were obtained by use of the AN10000 system and the Quantem FLS program (Link Systems) based on the Hall continuum method for quantitation of X-ray spectra [1,8]:…”

“…In order to immobilize highly diffusible elements in a defined functional state of interest without redistribution or loss by preparation procedures the cells are converted into the solid state by cryofixation. The preparation of freeze-dried 100 nm thick cryosections has proved to be the most versatile method with respect to X-ray microanalytical quantitation, detection limit and spatial resolution [1][2][3]. Thus, studying cryosections of cells by X-ray elemental mapping appears to be a consequent technical improvement [4][5][6][7].…”