A.M. Stadhouders

Surface roughness and the use of a peak to background ratio in the X-ray microanalysis of bio-organic bulk specimens

The use of a net peak intensity and of a peak to background (P/B)‐ratio of sulphur and chlorine is examined in the X‐ray microanalysis of a 2·4% w/w S bulk standard in Spurrs epoxy resin. In calculating the P/B‐ratio, the background intensity is calculated for the same energy region as for the net peak. Analyses were carried out on the flat top of the standard and on the slope running down from the top on the side not facing the X‐ray detector. The results obtained for the peak to local background ratios from the top and the slope yielded a relatively small mean deviation (11%) while net peak intensities ultimately were reduced to 7% or less of the initial value for the flat top. This indicated that a peak to local background ratio is to be preferred in the quantitative analysis of bulk specimens which have poorly defined local tilt and takeoff angles. A second advantage is the inherent correction for beam current fluctuations.

X-ray microanalysis of picoliter microdroplets: Improvement of the method for quantitative X-ray microanalysis of samples of biological fluids

Abstract A new practical approach to quantitative X-ray microanalysis of very small volumes of biological fluids, combining wavelength dispersive analysis and the use of thin films as supports is presented. The results from this technique are compared with those obtained using earlier methods and were found as predicted from theoretical considerations to be very accurate. Because of the favourable ratios between the detected characteristic radiation and the background radiation coupled with the resultant shorter periods of analysis, the use of this new technique of X-ray microanalysis for liquid samples not only saves time, but also smaller elemental concentrations can be detected. Suitable procedures to subtract background radiation are presented and discussed.

Quantitative electron probe X-ray microanalysis of thin objects using an independent mass determination method

Abstract Elemental mass fraction determinations on thin biological specimens by quantitative electron probe X-ray microanalysis commonly use a total mass estimation based on registration of X-ray continuum. We have tested whether the accurary of such determinations on individual biological objects can be improved when a densitometric method for mass determination is used. Air-dried femtoliter droplets with dry masses in the order of 10−16 kg were used as model objects. An improvement of over two-fold upon the continuum method could be achieved.

Quantitative electron probe x-ray microanalysis and densitometric mass determination of individual rat blood platelets

An improved method for quantitative electron probe X-ray microanalysis of thin biological specimens, introduced recently, has been applied to the elemental analysis of rat blood platelets. The method uses the X-ray signal to quantify the elemental content of an object and electron scattering to determine the total dry mass of the object. Along with the dry mass distribution, data were obtained on the content and mass fraction of calcium, magnesium, and sulfur in 31 individual platelets. The mean platelet dry mass was found to be 985 fg. The mean Ca, Mg, and S contents were 1.80, 3.41, and 18.3 fg, with mean dry mass fractions of 0.195, 0.396, and 1.96%, respectively. Furthermore, these elements appear to be unevenly distributed among the platelet population.

Calibration of transmission electron microscopic mass determination using objects with known mass distribution

An improved calibration procedure is described for dry mass determination methods, that are based on microdensitometry of transmission electron micrographs. Polystyrene latex spheres, that are supposed to have a homogeneous mass distribution, are used for the calibration procedure. By computer fitting of the theoretically expected mass distribution to the distribution measured in a line through the centre of a sphere, a mass calibration factor is obtained. The number of measurements required for a predetermined calibration accuracy can be decreased considerably as compared to other calibration methods. Accurate calibration and reproducible adjustment of the electron optical magnification are required.

Elemental analysis of individual rat blood platelets by electron probe X-ray microanalysis using a direct quantification method

SummaryThe elemental content of individual rat blood platelets and their dense granules was determined by electron probe X-ray microanalysis using a direct quantification method with microdroplets as standards. The quantification procedure was a modification of the ‘direct mass’ method involving a correction for differences in electron beam intensity in the analysis of standards and specimens. Whole air-dried platelets has a mean magnesium content of 12·10−8 nmol (SE=1·10−8 nmol, n=68) and a mean calcium content of 3.2·10−8 nmol (SE=0.3·10−8 nmol, n=68). A good correlation was found between the magnesium content and the phosphorus content of the dense granules of the platelets (r=0.95). There was also good correlation (r=0.77) between the number of dense granules per platelet and the magnesium content of the platelets.

The use of backscattered electrons in analytical electron microscopy for the measurement of the mass of individual rat blood platelets

SummaryThe backscattered electron signal, generated in individual cells, has been used to measure the dry mass of these cells. Absolute mass values were obtained by comparing the backscattered electron signals of cells to the signals of polystyrene-latex spheres of known mass. The technique was carried out in an automated analytical scanning transmission electron microscope and applied to rat blood platelets.The resulting mass distributions agreed well with the distribution measured with a method that uses the transmitted electron signal by means of densitometric analysis of electrographs. Also the range of masses was in agreement with values deduced from data in the literature.The fully automated technique has the advantage that it is direct, fast, and that thicker specimens can be measured than is possible using the transmitted electron signal. The method is intended for use in combination with quantitative electron probe X-ray microanalysis and is then able to produce elemental mass fractions of biological specimens at the subcellular level.

Simultaneous use of energy-dispersive and wavelength-dispersive spectrometers in X-ray microanalysis—development of an adjustable aperture in front of the energy-dispersive spectrometer

Abstract An adjustable aperture in front of the energy-dispersive spectrometer was developed which allows a mechanical lowering of the spectrometer sensitivity. A simultaneous operation of the energy-dispersive and the wavelength-dispersive spectrometers, with optimal spectrometer resolution and moderated dead time (

Simple procedure for the production of carbon‐polymer support grids for use in X‐ray microanalysis

A simple and rapid procedure for the production of low‐cost carbon‐polymer support grids is described. These grids are particularly useful for X‐ray microanalysis of thin specimens because of their low X‐ray background properties. The grids can be prepared for immediate use, which guarantees constant quality.