A method for preparing absolute standards for quantitative calibration and measurement of section thickness with X‐ray microanalysis of biological ultrathin specimens in EMMA

Abstract

Specimen thickness is determined using X-ray microanalysis in conjunction with atomic absorption spectrophotometry. Specimens of evaporated metal films and standards comprising fixed concentrations of element (such as potassium) in organic media are used to determine elemental concentrations of endogenous elements in embedding resin, from which section thickness can also be determined. The method finally depends only on the single measurement of X-ray emission of such an element in the specimen. Calibration of the X-ray detector over the energy range with mineral salts enables the standard to be used for the quantitative analysis of any element under investigation.

Sub-second quenched-flow/X-ray microanalysis shows rapid Ca2+ mobilization from cortical stores paralleled by Ca2+ influx during synchronous exocytosis in Paramecium cells

“…In addition, we controlled [Ca] in the Ca bead standards by atomic absorption or neutron activation (data not shown). As confirmed by these two independent physical methods, the spot mode analysis allowed us the calibration of the Ca Ka counts and a direct indication of [Ca] values per unit volume (rather than per kg dry weight) of subcellular structure (Chandler, 1976;Dörge et al, 1978;Hall, 1979). Effects of osmium (mainly fluorescence), though small (< 25%), were corrected based on an experimentally determined correction factor (Hardt and Plattner, 1999).…”

“…For resting or AED-stimulated cells, net Ca Ka count integrals registered over any subcellular structure were pooled to determine mean values and standard errors (n, N number of sites or cells analyzed), as established by Chandler (1976) and Dörge et al (1978).…”

“…Two widely different types of external standards were used for calibration: (i) organic Ca-compounds included in Spurrs resin (Chandler, 1976) purchased from British BioCell (Cardiff, Wales) and distributed by Plano (Wetzlar, Germany), and (ii) Ca-exchange beads incubated at different [Ca 2 ] (DeBruijn, 1981). Method (i) was used since it is not liable to Ca loss, while standards from method (ii) served to analyze any potential side-effects (like Ca loss or diffusion) since beads can be processed to sections in the same way as paramecia.…”

Quantitative Energy-Dispersive X-Ray Microanalysis of Calcium Dynamics in Cell Suspensions during Stimulation on a Subsecond Time Scale: Preparative and Analytical Aspects as Exemplified with Paramecium Cells

“…We used two types of standards. (i) Organic Ca compounds at different concentrations in epoxide resin according to Chandler (1976;Dorge et al, 1978) were obtained from British BioCell (Cardiff, Wales) or PIano (Wetzlar, Germany); (ii) Ca-exchange (Chelex) beads, according to DeBrujin (1981), incubated at different [Ca 2 +), were a gift from Bio-Rad , with KF present or absent during freeze-substitution, in two independent experiments (10 structures each collected from 10 cells). Normalized data (CaK a signal = 100% for samples freeze-substituted in the presence ofKFJ reveal considerable signal loss from alveolar sacs (as) and cytoplasm (cy) when samples are processed without KF in the freeze-substitution medium.…”

“…Since this rather strict correlation of both signals also clearly excludes any significant Ca 2 + leakage into surrounding embedding medium, we are confident about the reliability of recordings in cells, where most of the Ca is bound to proteins. Figure 9 documents calibration with sections from two types of standards (designated types I and ID: (i) epoxy resin with Ca 2 + directly included at different concentrations (Chandler, 1976) and (iD Ca 2 +-impregnated Chelex beads as described above. A priori, Ca 2 + loss cannot be a significant problem with type I standards, which, thus, can serve as addi- tional controls.…”