Quantitative X-ray mapping of 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.

Elemental imaging by EELS and EDXS in the analytical electron microscope : Its relevance to trace element research.

Electron energy loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDXS) can be used to obtain elemental maps from thin biological samples in the analytical electron microscope. The EELS is particularly sensitive for the low-atomic-number elements, including C, N, and O, as well as other elements with favorable ionization cross-sections, such as Fe. The EDXS is useful for a complementary range of atoms, such as P, S, K, and Ca. A system is described for obtaining elemental distributions in an analytical electron microscope operated in the scanning transmission mode at 100-200 keV beam energy. The spatial resolution is typically limited to 10-20 nm when a conventional source is used. A satellite microcomputer controls acquisition of EELS and EDXS data from successive pixels in an image. These data are processed "on-the-fly" by a host computer to remove the noncharacteristic background intensity. Resulting images are stored on disk and can be analyzed by means of an image display system controlled by interactive software. The technique is demonstrated with elemental maps from two samples: alveolar macrophages containing respirable particles; and pancreatic beta cells that secrete insulin.

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 microns 3) 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: our microanalysis cell preparation method preserves whole intact lymphocytes; there is no systematic difference in results from the two methods of analysis; electron probe X-ray microanalysis can determine the variation in magnesium concentration among individual cells.

On the use of ionization cross sections in analytical electron microscopy.

There are two approaches to the utilization of the ionization cross section, Q, for use in the determination of kappa AB factors for quantitative microanalysis in the analytical electron microscope. The first approach is to interpolate a value of Q from experimentally determined kappa AB factors at a fixed accelerating voltage (kV). The second approach uses a theoretical parameterization of Q generated by fitting the fundamental Bethe expression to selected experimental values of Q over a wide range of kV. This paper discusses the relative merits of the two approaches.

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 samples 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 (less than or approximately 100 nm diameter) are used.

Imaging of calcium and aluminum in neurofibrillary tangle-bearing neurons in parkinsonism-dementia of Guam.

We report the distribution and imaging of calcium and aluminum in neurofibrillary tangle (NFT)-bearing neurons within Sommer's sector of the hippocampus in Guamanian patients with parkinsonism-dementia, using a method of computer-controlled electron beam x-ray micro-analysis and wavelength dispersive spectrometry. Calcium and aluminum were distributed in cell bodies and axonal processes of NFT-bearing neurons. The elemental images show that both calcium and aluminum deposits occur within the same NFT-bearing hippocampal neuron in this dementing disease, suggesting that these elements are involved in NFT formation. No prominent concentrations of calcium and aluminum were imaged in non-NFT-containing regions within the pyramidal cell layer of the parkinsonism-dementia cases or in the control cases. These findings support the hypothesis that secondary hyperparathyroidism resulting from low environmental calcium and magnesium in the high-incidence focus of amyotrophic lateral sclerosis and parkinsonism-dementia on Guam had led to abnormal deposition of calcium and aluminum in the central nervous system.

Keratopathy associated with intracorneal glass.

A progressive nonedematous keratopathy developed in a 36-year-old patient after she was struck in the eye by glass fragments. Biopsy material that was examined by electron microscopy and electron beam microanalysis demonstrated the presence of intracorneal glass fragments, which could not be detected clinically. Retained intracorneal glass, generally thought to be completely inert, can be associated with a chronic keratopathy.

Artifacts in energy dispersive x-ray spectrometry in the scanning electron microscope (II).

The quality of x-ray spectra obtained with an energy dispersive x-ray spectrometer on an electron beam instrument can be severely compromised by the presence of electromagnetic interference. Sources of electromagnetic interference include ground currents and signals generated by time-varying currents in instrument components such as scan coils. Spectrometer resolution can be degraded by the accumulation of ice and vaccum oil on critical components of the device. Operation at high electron energy can cause artifacts in spectra due to direct entry of electrons and spurious x-rays into the detector. Processing high energy photons (above 40 keV) can lead to detector saturation effects which degrade resolution and affect dead time correction. Transmission of high energy x-rays through the detector accompanied by Compton scattering can lead to a distortion of the low energy portion of the spectrum.