Suggestions for the quantitative X-ray microanalysis of thin sections of frozen-dried and embedded biological tissues

When a microregion in a thin section of frozen-dried and embedded tissue is analysed by the conventional electron-probe X-ray continuum-normalization method, the measured quantity is in mmol of element per kg of embedded specimen. As each microregion contains an unknown amount of embedding medium, this quantity generally lies indeterminately somewhere within the wide range between mmol of element per kg of hydrated tissue and mmol of element per kg of dehydrated tissue. However, if a ‘tag’ element is incorporated in the embedding medium, the contribution of the medium to the local continuum count in each probed field should be measurable, and the X-ray data may then unambiguously yield mmol of element per kg of dehydrated tissue. This result should not be affected by shrinkage on freeze-drying or by incomplete replacement of water by embedding medium. The same X-ray data can additionally provide estimates of mmol of element per unit volume, mmol of element per kg of hydrated tissue and local dry-mass fraction. However, these estimates are subject to errors due to tissue shrinkage, incomplete replacement of water and beam damage.     

The effect of aluminium coating on elemental standards in X-ray microanalysis

The standardisation of frozen hydrated bulk biological specimens using gelatin standards is described. The relationship between corrected elemental X-ray counts and ionic concentration was found to be linear, and minimum detectable limits for each element are stated. Variations in uncorrected standard curves were found to be due to changes in aluminium coating thickness. There was an inverse relationship between coating thickness and elemental X-ray counts. The factors causing this are discussed. To avoid errors arising from inconsistent aluminium thickness, experimental material should only be compared with standards of similar aluminium net counts. This can be achieved most easily by mounting and analysing specimen and standard together.     

Determination of gold-labelled surface receptors on single cells by X-ray microanalysis

A standardless X-ray microanalytical procedure has been developed to determine the number of gold-labelled surface receptors on whole single cells. The effect of the injection of K₂PtCl₄ into mice on gold-labelled concanavalin A (Con A) receptors on peritoneal macrophages was examined with an energy dispersive X-ray detector in an SEM. The numbers of gold particles seen in electron micrographs and estimated by fluorescence photometric measurements of fluorescein isothiocyanate-labelled Con A receptors were correlated with the X-ray microanalytical results.     

Evaluation of several common standards for the X-ray microanalysis of thin biological specimens

The question of the best type of standard to use for X-ray microanalysis of thin biological specimens remains unanswered. Standards embedded in an organic matrix have the advantage that they resemble biological specimens, but their composition is generally not known exactly. We compared several standards and, surprisingly, inorganic binary salts sprayed onto a supporting film were the most suitable: they corresponded closely with several other methods using organic matrices; they were easily produced; and their composition is known. Glutaraldehydeurea aminoplastic resin thin sections and thin films containing dissolved salts were problematic. The composition of the polymer appears to be variable, and the thin films did not correspond with any other standard tested. Chelex¹⁰⁰ bio-standard beads and flakes loaded with accurately determined concentrations of ions, embedded in epoxy resin and thin sectioned, tended to correspond to the results obtained with the binary salts. However, the results from some bio-standards were inexplicably aberrant. An epoxy resin standard was used for bromine, and was found to agree closely with the binary standards.     

Quantification for the X-ray microanalysis of cryosections

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.     

Beryllium coating for biological X-ray microanalysis

Beryllium is ideal for coating biological specimens for light element X-ray microanalysis at low temperature. It has higher electrical and thermal conductivity at 100 K and lower absorption of X-rays of biological interest than carbon, aluminium or chromium. It produces no detectable characteristic X-rays. When adequate precautions are taken beryllium is a valuable alternative to other coating materials. Because of its toxicity, however, it should not be used indiscriminately.     

Quantitative study of spurious X-ray production in thin film microanalysis

When X-ray microanalysis is performed in a TEM on a thin area of a specimen, some a priori indistinguishable spurious photons produced in other zones of this specimen are always recorded. Several mechanisms contribute to this production. For instance, some Bremsstrahlung and characteristic photons are generated by secondary and Auger electrons; a conservative upper bound to this particular contribution is calculated for several materials, and the present approach is compared to the Monte Carlo simulation. It is then shown that, in special test-specimens, the total extraneous contribution of the thick parts of the specimen to the spectra recorded in a thin zone can be measured; different instruments may now be compared in this respect. In the HB5 STEM, this total contribution remains low; its main cause is beam scattering in the specimen, not before it. Finally, an experimental procedure for estimating this bulk contribution in any specimen of interest is proposed. Calculations and experiments are illustrated for the case of gallium arsenide.     

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.     

Understanding thin film X-ray spectra

Formulae suitable for predicting X-ray production cross-sections in thin foils are described. A modified Bethe-Heitler equation seems well suited to describe the production of bremsstrahlung photons and, for fixed experimental conditions, may be expressed in a simple parametric form. Simple equations for calculating characteristic photon production are less satisfactory and the predictions of different formulae differ markedly. For microanalytical purposes, however, where relative rather than absolute cross-sections are required, the differences become less pronounced. Finally, some uses for the cross-sections in X-ray microanalysis are considered.     

Agar standards for quantitative X-ray microanalysis of resin-embedded plant tissues

Calibration standards for quantitative X-ray microanalysis of resin-embedded plant tissue were prepared by adding 6600 mM KC1 to 5% agar. Agar blocks with an edge length of 1–2 mm were rapidly frozen, freeze-dried and embedded in styrene-methacrylate. Dry sections 1 μm thick were mounted on adhesive-coated grids. Apart from fine-scale inhomogeneities caused by ice crystal formation, the KC1 is evenly distributed in the agar blocks. The peak-to-continuum values of K and Cl were highly linearly correlated to the K and Cl contents over the whole concentration range.     

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

A system based on a personal computer has been developed which provides a relatively inexpensive way to equip an electron microscopy laboratory for quantitative elemental analyses of cryosectioned biological samples. This system demonstrates the feasibility of making an X-ray analyser from a personal computer, together with commercially available hardware and software components. Hardware and software have been assembled to drive the beam in a scanning electron microscope, collect and analyse X-ray spectra, and save, retrieve, and analyse data. Our software provides a menu-controlled user interface to direct spectra acquisition and analysis. Spot analyses, video images, and quantitative elemental images may be obtained and results transferred in ASCII format to other computers. Wet weight, as well as dry weight, concentrations are calculated, if measurements were made of areas of the hydrated sample before it was freeze-dried. Grey-level copies of video and quantitative elemental images may be made on a laser printer.     

Ionic composition of rat airway surface liquid determined by X-ray microanalysis

The thin layer of liquid that lines the conducting airway epithelium, the airway surface liquid (ASL), is important for mucociliary clearance. Altered ionic composition and/ or volume of the ASL play a major role in the pathology of airway diseases such as cystic fibrosis. Since the ASL is a thin layer, it has been difficult to exactly determine its composition. The present paper describes two techniques that have been developed and used to study ASL composition: X-ray microanalysis of frozen hydrated rat trachea, and an ion-exchange (dextran) bead method, where dextran beads were placed on the airway epithelium to equilibrate with the ASL; the beads were then collected under silicone oil, dried and analyzed by X-ray microanalysis. The results from both frozen-hydrated specimens and from the dextran beads showed that ASL from rat trachea is hypotonic. Concentrations of Na, P, S, and K were higher in the frozen-hydrated ASL, in which mainly the mucus layer is analyzed, compared with the dextran bead method, in which mainly the periciliary liquid is sampled. Also the composition of rat nasal fluid was investigated by the dextran bead method. This fluid was somewhat hypertonic because of a high K concentration. The ionic composition of the nasal and tracheal fluid can be manipulated by cholinergic or alpha- or beta-adrenergic stimulation. Collecting ASL with dextran beads did not disturb the integrity of the airway epithelium. The ionic composition of the collected beads remained stable for several days during storage in silicone oil. It is concluded that X-ray microanalysis is a suitable method to determine the ionic composition of ASL.     

The accurate alignment of energy-dispersive X-ray detectors for quantitative microanalysis

Modern collimator design for energy-dispersive X-ray detectors requires very accurate positioning of the crystal/collimator assembly in order to achieve the maximum solid angle of collection for the irradiated volume on the specimen. Thus it is important to have a method of checking the alignment of the detector when mounted on the microscope and under vacuum. This paper describes a number of techniques, principally X-ray mapping, for performing such an alignment check. These techniques are applicable to windowless detectors as well as to those with integral windows which will support atmospheric pressure. Methods of obtaining the non-standard modes of microscope operation suitable for this task are described, and some suggestions are made for ways of moving the crystal/collimator assembly and monitoring this movement while it is in progress.     

Limitations in the X-ray microanalysis of thin foils in a scanning transmission electron microscope

The X-ray microanalysis of thin foils has been investigated using a scanning transmission electron microscope fitted with an energy dispersive spectrometer. Thin foils prepared from an iron-nickel and an aluminium-zinc-magnesium-copper alloy have been observed and analysed. For foil thicknesses between 200 and 300 nm the X-ray intenstiy ratios are consistent with X-ray absorption characteristics. For regions less than 200 nm thickness the measured intensity ratios increase by up to a factor of 5. These results have been explained in terms of a solute enriched or depleted surface layer developed during the preparation procedure.     

The use of adhesive-coated grids for the X-ray microanalysis of dry-cut sections in the TEM

Sections cut dry for the X-ray microanalysis of diffusible elements were fixed to adhesive-coated single fine-bar grids. The drawbacks of folding grids normally used for this purpose can thus largely be avoided.     

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

A series of computer programs have been written for use in a multi-user resource center for biological microanalysis. They are tied together by a “Main Menu” program which acts as a traffic director and guides the investigator into whatever option is desired. These programs have been written with very clear instructions and interaction points. As a result, a complex handbook of options and responses is not required. Consquently, almost no training is required to use these programs as they are essentially self-explanatory. Extensive error checking has been included so that most errors are identified and corrections are requested without causing a program halt. Options available to the user provide for selected “region” analyses, elementally quantitative image analysis, and sorting of the resulting data files. A complete computer code printout is included in this report.     

Quantitative EDS and WDS X-ray microanalysis of semiconductor materials: Principles and comparisons

The basic principles and procedures for performing accurate X-ray microanalyses on semiconductor materials as well as the differences and advantages of EDS (energy-dispersive spectroscopy) and WDS (wavelength-dispersive spectroscopy) are presented. Many of the techniques discussed are the result of extensive experience with some photovoltaic materials such as CuInSe₂, GaAs, CdTe, CdS, CdSnP, InP, ZnP, and amorphous Si-based alloys. Results of an experiment designed to compare the performance of an energy-dispersive X-ray spectrometer (EDS) and a wavelength-dispersive X-ray spectrometer (WDS) on some semiconductor materials are presented. CdTe, CdSe, CuInSe₂, ZnSe, and InSb standards were used to measure the k-ratios with pure element standards employing both types of X-ray spectrometers to demonstrate the ability of each to measure elemental compositions under various operating conditions. Both types of spectrometers were utilized on a Cameca MBX electron microprobe at identical X-ray take-off angles. The EDS spectrometer was found to perform equal to the WDS spectrometer when integral peak-to-background ratios were greater than seven, when the X-ray line being measured was greater than about 1,000 eV, and when adjacent peaks were separated by more than three times the detector energy resolution.     

Inhomogeneity in epoxy resin standards for quantitative biological X-ray microanalysis

It is shown by X-ray point analyses and line scans that the concentrations of sodium and potassium, as crown ether complexes, in epoxy resin may not be of uniform distribution. The concentrations may be substantially higher in a thin layer at the base of the block. It is recommended that chemical analysis of a selected central region of a block, not the intact block, be carried out to establish the true concentration. This may be substantially lower than the nominal concentration. This problem appears to be less acute with cryptate complexes of sodium and potassium but a similar trend is nevertheless apparent.     

X-ray microanalysis of cultured keratinocytes: methodological aspects and effects of the irritant sodium lauryl sulphate on elemental composition

Irritant substances have been shown to induce elemental changes in human and animal epidermal cells in situ . However, skin biopsies are a complicated experimental system and artefacts can be introduced by the anaesthesia necessary to take the biopsy. We therefore attempted to set up an experimental system for X-ray microanalysis (XRMA) consisting of cultured human keratinocytes. A number of methodological aspects were studied: different cell types, washing methods and different culture periods for the keratinocytes. It was also investigated whether the keratinocytes responded to exposure to sodium lauryl sulphate (SLS) with changes in their elemental composition. The concentrations of biologically important elements such as Na, Mg, P and K were different in HaCaT cells (a spontaneously immortalized non-tumorigenic cell line derived from adult human keratinocytes) compared to natural human epidermal keratinocytes. The washing procedure and time of culture influenced the intracellular elemental content, and rinsing with distilled water was preferred for further experiments. Changes in the elemental content in the HaCaT cells compatible with a pattern of cell injury followed by repair by cell proliferation were seen after treatment with 3.33 µ m and 33 µ m SLS. We conclude that XRMA is a useful tool for the study of functional changes in cultured keratinocytes, even though the preparation methods have to be strictly controlled. The method can conceivably be used for predicting effects of different chemicals on human skin.     

Methods for determining the composition of nasal fluid by X-ray microanalysis

The nasal fluid is an easily accessible form of airway surface liquid. The objective of this study was to find a technically easy and reproducible method for sampling and analysis of this fluid. In a pilot study, several methods to carry out X-ray microanalysis of sub-microliter droplets were compared. Acceptable results were obtained with several of these methods (pipeting on filter paper or analysis of frozen-hydrated droplets at low temperature). Nasal fluid was collected from the inferior turbinate with a micropipette after occlusion of a nostril for 5-10 minutes. Ion concentrations in nasal fluid from six control subjects were (in mM, mean +/- standard error): sodium (Na) 127 +/- 6, chloride (Cl) 140 +/- 7, potassium (K) 27 +/- 3, and calcium (Ca) 5 +/- 1. This sampling method proved difficult to apply to cystic fibrosis (CF) patients because of the viscous quality of their nasal secretion. Therefore, an alternative method was devised. Sephadex G-25, ion exchange beads were mounted on double-sided tape, which was stuck on a filter paper as support. The filter paper was applied for 10 minutes to the nostril of a subject, and kept loosely in place. During the exposure period, the nasal fluid equilibrates with the beads. After removal of the filter paper with the beads from the nostril, the beads were rinsed with a hydrophobic volatile silicone oil to remove excess nasal fluid, dried, and analyzed. This method of collection is not cumbersome for the subject and gives results similar to those obtained by the direct collection method: Na 142 +/- 28 mM, Cl 150 +/- 36 mM, K 43 +/- 10 mM (mean and standard error of four determinations). Small differences between the filter method and the bead method can be explained by the fact that the filter method measured total nasal fluid, whereas the bead method measures predominantly the fluid component. Subjects suffering from mild respiratory illness or rhinitis had higher values for Na, K, and Cl in their nasal fluid.