Carol R. Swyt

Separation of overlapping core edges in electron energy loss spectra by multiple-least-squares fitting

A multiple-least-squares fitting procedure for quantitating electron energy loss spectra is demonstrated on some strongly overlapping core edges. The method, first applied by Shuman and Somlyo [Ultramicroscopy 21 (1987) 23], takes into account plural inelastic scattering and can be applied under conditions of non-uniform sample thickness where Fourier deconvolution techniques are invalid. By using appropriate reference spectra generated from pure compounds, quantitation of potassium and calcium (L23 edges) is possible in the presence of carbon (K edge), and sulfur in the presence of phosphorus (L23 edges). Some of the advantages and limitations of the multiple-least-squares approach are discussed.

Mass thickness determination by electron energy loss for quantitative X-ray microanalysis in biology

As is well known, electron energy loss spectroscopy can be used to determine the relative sample thickness in the electron microscope. This paper considers how such measurements can be applied to biological samples in order to obtain the mass thickness for quantitative X‐ray microanalysis. The important quantity in estimating the mass thickness from an unknown sample is the total inelastic cross section per unit mass. Models for the cross section suggest that this quantity is constant to within ±20% for most biological compounds. This is comparable with the approximation made in the continuum method for measuring mass thickness. The linearity of the energy loss technique is established by some measurements on evaporated films and quantitation is demonstrated by measurements on thin calcium standards. A significant advantage of the method is that the energy loss spectrum can be recorded at very low dose, so that mass thickness determination can be made before even the most sensitive samples suffer damage resulting in mass loss. The energy loss measurements avoid the necessity to correct the continuum measurement for stray radiation produced in the vicinity of the sample holder. Unlike the continuum method the energy loss technique requires uniform mass thickness across the probe area, but this is not usually a problem when small probes (<100 nm diameter) are used.

Quantitative X-ray mapping biological cryosections

The potential for applying X-ray mapping to the elemental microanalysis of biological cryosections is discussed. Methods are described for acquiring and processing data, including use of the top-hat digital filter to remove the average effects of the background contribution. Practical considerations for X-ray mapping are discussed in terms of typical counts per pixel and minimum detectability which depends on the number of pixels chosen to integrate the signal. These aspects are illustrated with elemental maps (Na, P, K, Ca and Fe) from freeze-dried cryosections of mouse cerebellar cortex. A calcium sensitivity in the range 0.5 to 2.5 mmol/kg wet weight of tissue is demonstrated. The correction for overlap of potassium K beta and calcium K alpha is demonstrated with X-ray maps from cryosectioned synaptosomes of squid optic lobe. Quantitative results obtained using internal standards to determine wet weight concentrations are in reasonable agreement with expected values. Alternate schemes applicable to X-ray maps for determining the dry mass concentration, such as the peak/continuum (Hall method), are also discussed.

Combined elemental and stem imaging under computer control

Abstract A computer-controlled analytical electron microscope has been used to combine digitally acquired elemental maps and STEM images. Real time processing of the electron energy loss and energy-dispersive X-ray signals allows an accurate subtraction of the spectral background at each pixel. The resulting images reflect the true elemental distribution rather than mass thickness variations in the sample. Acquisition of several signals one pixel at a time allows elemental distributions and morphology to be correlated.

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 Burkitts 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.