Standards for the application of X-ray microanalysis to biological specimens

A review on the subject of compounds used as standards for biological X-ray microanalysis is presented. The general approach used for standardization has been to use standards which resemble the specimen closely in composition. Thus, standards based on proteins have been used for analysis of quench-frozen cryosectioned specimens, whereas standards based on embedding resins have been used for resin-embedded material. The properties of, and problems associated with, each type of standard are recognized and have been well documented. The choice and analysis of standard should not be a drawback to fully quantitative analysis of biological material. Attention is drawn to the fact that the problems associated with any quantification procedure need to be kept in mind when analysis of standards is undertaken.     

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g , washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m m ammonium acetate, 300 m m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.     

A sample preparation for quantitative determination of magnesium in individual lymphocytes by electron probe X-ray microanalysis

We present a sample preparation method for measuring magnesium in individual whole lymphocytes by electron probe X-ray microanalysis. We use Burkitt's lymphoma cells in culture as the test sample and compare X-ray microanalysis of individual cells with atomic absorption analysis of pooled cell populations. We determine the magnesium peak-to-local continuum X-ray intensity ratio by electron probe X-ray microanalysis and calculate a mean cell magnesium concentration of 39± 19 mmol/kg dry weight from analysis of 100 cells. We determine a mean cell magnesium concentration of 34 ±4 mmol/kg dry weight by atomic absorption analysis of pooled cells in three cell cultures. The mean cell magnesium concentrations determined by the two methods are not significantly different. We find a 10% coefficient of variation for both methods of analysis and a 30% coefficient of variation in magnesium concentration among individual cells by electron probe X-ray microanalysis. We wash cells in ammonium nitrate for microanalysis or in buffered saline glucose for atomic absorption analysis. We find cells washed in either solution have the same cell viability (85%), recovery (75%), cell volume (555 μm³) and cytology. We air dry cells on thin film supports and show by magnesium X-ray mapping that magnesium is within the cells. We conclude that: (a) our microanalysis cell preparation method preserves whole intact lymphocytes; (b) there is no systematic difference in results from the two methods of analysis; (c) electron probe X-ray microanalysis can determine the variation in magnesium concentration among individual cells.     

The continuum normalization method for quantification of X-ray spectra in biological microanalysis. 1. Generalized bremsstrahlung production cross-sections and analysis using standards

The thin self-supporting biological specimens used for quantitative X-ray microanalysis are problematical because the sections are most unlikely to be uniform in thickness or density, so the intensities of the characteristic lines alone are not a good measure of composition. The method developed to overcome these problems was introduced by T. A. Hall in 1971 and uses the bremsstrahlung or continuum intensity recorded in the X-ray spectrum to normalize each characteristic line, and hence is frequently referred to as the continuum normalization (CN) procedure.
  Reformulating the CN method of quantification in terms of generalized cross-sections and calculating more accurate values of bremsstrahlung production using a formula allows us a better understanding of the options open to the analyst of biological thin sections by which the errors in the measurement may be reduced. If one chooses to use the original Hall (1971 ) method using Kramers cross-sections, the window measuring the continuum for normalization should be set in the 4–7 keV region for typical scanning electron microscope and microprobe beam energies, 20–40 kV, and above 10 keV for transmission electron microscope energies of 80 kV and above. Although it is clear that peak counts must not contribute to the white count, the window should be as wide as possible to reduce statistical errors.     

Study of potassium deficiency in cardiac muscle: quantitative X-ray microanalysis and cryotechniques

An imbalance of potassium in cardiac muscle causes an alteration of heart function. The distribution and concentration of potassium in rat papillary heart muscle was studied using cryofixation and X-ray microanalysis. Freeze-dried cryosections and sections of freeze-dried, embedded tissue were analysed. Bulk frozen specimens were freeze-dried either in a vacuum or by a new technique using liquid propane as a cryodehydration medium. These two methods of freeze-drying were tested for elemental retention in other specimens, with comparable results. A potassium concentration of 120 mmol/l was measured in normal myocytes of cardiac papillary muscle compared to 80 mmol/l in myocytes of animals stressed by a temperature of 45°C for 1 h. The presumed physiological significance of the findings is discussed.     

Improved quantitative electron probe X-ray microanalysis of biological cryosections using an EDS germanium detector and a super-atmosphere thin window (SuperATW)

A new Link energy-dispersive GEM detector with SuperATW window was tested for quantitative electron probe microanalysis of low calcium and sodium concentrations ([Ca], [Na]) in intracellular compartments of cardiac myocytes. We compare Ca profiles with high count statistics and similar peak area collected under the same conditions with either a Be-windowed Si and a Ge SuperATW detector. The height of the Ca peak was increased by 7%, the full width at half-maximum height was reduced by 9% with the Ge detector. The counts statistics of the Ca Kα peak improved by 9% and the partial overlap with the K Kβ peak was better deconvoluted. We calculate [Ca] and errors of the single measurement in mitochondria of guinea-pig cardiac myocytes from spectra acquired with a Si or Ge detector. For identical analysis conditions, the [Ca] were identical; however, with the Ge SuperATW detector, the calculated error of the single measurement was only 1/2.7 of that calculated from measurements with the Si detector. We compare the peak area of identical [Na] in spectra collected with the Be-windowed Si detector and Ge SuperATW detector. The peak area was significantly higher with the SuperATW Ge than with the Si detector and Be window, whereas the continuum in the range 4–10 keV was comparable, demonstrating the improved sensitivity for low atomic elements such as Na of the Ge SuperATW detector. [Na] and errors of the single measurement in mitochondria of quiescent guinea-pig cardiac myocytes were calculated from spectra acquired with the Si or the Ge detector. The use of the Ge SuperATW detector improved the detectability limit for sodium by more than 80% and reduced the error of the single measurement by a factor of 7–8.     

Absorption microanalysis with a scanning soft X-ray microscope: mapping the distribution of calcium in bone

This article describes the scanning transmission X-ray microscope operated at the National Synchroton Light Source. The application of the instrument to elemental analysis is detailed. In particular, qualitative results on the calcium distribution in human skull tissue are presented.     

Quick sampling and perpendicular cryosectioning of cell monolayers for the X-ray microanalysis of diffusible elements

A quick sampling and preparation method for freezing of cell monolayers is described. The cells are grown on a large Formvar film supported by a frame of polystyrene. A polyvinylpyrrolidone (PVP) solution is applied to one side of the film forming a flat disc when frozen with a pair of pliers precooled in liquid nitrogen. The PVP solution provides the specimen with sufficient strength and may be used as an elemental standard for absolute quantification if salts of known concentrations are added. Manipulation of the cells prior to freezing is thus restricted to a minimum, which eliminates possible harmful treatments like scraping and centrifugation. The procedure is quickly performed, the freezing being completed within 30 s of the cells having been removed from the culture well. The analytical results reveal low and stable Na: K ratios. Our results confirm that cells in vitro are comparable to cells in vivo with respect to elemental composition.     

Development and comparison of the methods for quantitative electron probe X‐ray microanalysis analysis of thin specimens and their application to biological material

In recent years, there has been a return to the use of electron probe X‐ray microanalysis for biological studies but this has occurred at a time when the Hall programme which acted as the mainstay for biological microanalysis is no longer easily available. Commercial quantitative routines rely on the Cliff‐Lorimer method that was originally developed for materials science applications. Here, the development of these two main routines for obtaining quantitative data from thin specimens is outlined and the limitations that are likely to be met when the Cliff‐Lorimer routine is applied to biological specimens is discussed. The effects of specimen preparation on element content is briefly summarized and the problems encountered when using quantitative analysis on resin‐embedded materials emphasized.     

Calcium in secretory vesicles of neurohypophysial nerve endings: quantitative comparison by X-ray microanalysis of cryosectioned and freeze-substituted specimens

The calcium content of individual secretory vesicles in rat neurohypophysial nerve endings was measured by quantitative electron probe X-ray microanalysis. Directly frozen control and potassium-depolarized isolated endings were analysed using two presumably equivalent preparative techniques: (1) freeze-substitution in presence of oxalic acid followed by sectioning of resin-embedded pellets; or (2) direct cryosectioning of the frozen pellets followed by freeze-drying in the column of the microscope. In the pellets of stimulated endings, both approaches revealed an increase in the calcium content of neurosecretory vesicles. This increase was statistically more significant in the specimens prepared by cryosectioning, probably because in this case the contribution of 'dead' nerve endings could be eliminated on the basis of excessive cytoplasmic sodium and chloride. The results demonstrate that an increase in cytosolic calcium can lead to an increase in intravesicular calcium, and that when this occurs, it occurs within a subpopulation of vesicles in a given nerve ending. In addition, measured intravesicular calcium was dispersed over a wide range of concentrations, as predicted by the hypothesis of intravesicular calcium priming. When the vesicular calcium content was averaged per nerve ending, a relatively wide distribution of concentrations was again observed, indicating that some nerve endings respond more strongly to the stimulation than others.     

Skirting: a limitation for the performance of X-ray microanalysis in the variable pressure or environmental scanning electron microscope

The variable pressure or environmental scanning electron microscope (VP-SEM; ESEM) has become the microscope of choice for many scientists and technologists. Hence, the development of robust methods for X-ray microanalysis, limited by skirting, has become critical. In this paper, two pressure variation correction methods (Doehne and Gauvin) are compared. Both of these methods appear to be effective; the results were found to be well within 10% of the values obtained at 0 Pa. The Doehne method is dependent on an empirical factor (D), therefore the accuracy of the results will depend on the accuracy of this value. Also the Doehne method is compromised by the nonlinearity of the response with pressure. The Gauvin method is more user-friendly and more precise when considering the total range of pressure.     

Localization of Ca2+ -stores and tissue compartments with a Ca2+ -binding capacity in the organ of Corti of the guinea-pig by electron energy-loss spectroscopy

The addition of 10 mM CaCl₂ to glutaraldehyde fixative leads to the formation of small electron-dense deposits in the organ of Corti of the guinea-pig. These precipitates are mainly attached to cell membranes in contact with different extracellular lymphatic fluids. A higher number of precipitates is localized in the acellular parts of tectorial and basilar membrane. Electron energy-loss spectroscopy (EELS) was used to determine the elemental composition of the deposits formed. The spectra showed a prominent signal at the Ca²⁺ L_2,3 ionization edge. Oxygen could also be detected in all the precipitates analysed. EELS analysis of mitochondria of the inner and outer hair cells after conventional fixation (glutaraldehyde followed by post-fixation in OsO₄) revealed a small but significant calcium signal.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.