Standardization in biological analyses of aluminum: what are the needs?

A review of the literature of aluminum (Al) levels in biological samples, particularly Alzheimer's disease (AD) brain, reveals a lack of interlaboratory agreement at both the bulk and microprobe levels. One possible reason for this controversy may be the methods chosen for quantitation and standardization. Currently, the major problem affecting quantitation of Al at the bulk level is the lack of low-Al-concentration, matrix-matched certified standards. Although a number of certified aluminum bulk standards are available, most do not match well in matrix with the samples of interest. A similar situation exists for micro-probe standards in which commercially available pure metal foils and thin films of materials, such as metal oxides, are available but again do not match well in matrix. A review of the current status of quantitation of Al levels in biological analyses at the bulk and microprobe level is presented. Future directions to develop standards include the submission of currently used microprobe standards to a central laboratory for critical analysis and selection of the optimum standard, followed by establishment of interlaboratory and intertechnique comparisons. Other future directions include the adaptation of a standard protocol for the selection of tissue for analysis and criteria for data rejection, as well as the development of a standard system for data normalization and reporting.

"Standardless" quantitative electron probe microanalysis with energy-dispersive X-ray spectrometry: is it worth the risk?

"Standardless" procedures for quantitative electron probe X-ray microanalysis attempt to eliminate the need for standardization through calculation of standard (pure element) intensities. Either "first principles" calculations, which account for all aspects of X-ray generation, propagation, and detection, or "fitted standards" calculations, which use mathematical fits to measured intensities from a limited set of pure standards, can form the basis for standardless analysis. The first principles standardless analysis procedure embedded in the National Institutes of Health/National Institute of Standards and Technology comprehensive X-ray calculation engine and database, Desktop Spectrum Analyzer, has been tested against spectra measured on NIST standard reference materials, research materials, and binary compounds. The resulting distribution of errors is broad, ranging from -90% to +150% relative. First principles standardless analysis can thus lead to unacceptably large errors.

Trypanosoma cruzi: elemental composition heterogeneity of cloned stocks.

Concentrates of the epimastigote stage of Trypanosoma cruzi stocks derived from single cell clones and cultured under identical conditions display a spectrum of 'colors' varying from dark brown to milk white. The color of the concentrate is reproducible for a parasite stock. An essential component of the culture medium for epimastigote growth is hemin, an iron-containing compound. Consequently, it seemed possible that the color spectrum of the epimastigote stocks reflected differences in the uptake, concentration or utilization of iron. This report describes the quantitative studies utilizing electron probe X-ray elemental mapping, energy dispersive X-ray microanalysis, and energy dispersive X-ray fluorescence spectrometry of the epimastigote stage of two T. cruzi stocks (CA-I/72 and HO-3/15) which display extreme color differences. Striking and statistically significant quantitative differences were found in the levels of Fe, Zn, and K between the two stocks. On the basis of atomic ratios, the CA-I/72 stock contains approximately two-fold more Fe per cell than the HO-3/15 stock. However, in the case of Zn the ratio is reversed; the HO-3/15 stock contains approximately two-fold more Zn per cell than the CA-I/72 stock. The marked inter-stock differences which exist in the levels of several elements could modulate the pathogenicity, survival, or adaptability of T. cruzi and, consequently, be important factors in our understanding of the complex problem of Chagas' disease.

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.

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.

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.

Digital processing of electron energy loss spectra and images.

Processing in electron energy loss spectroscopy involves both data acquisition and analysis. The interface of an analytical electron microscope to a laboratory computer with a satellite microcomputer dedicated to data acquisition results in a system with a high degree of flexibility. In spectrum acquisition, channels may be selected around specific core edges, or dwell times may be varied continuously as a function of energy loss to reduce the dynamic range of the signal. Data transfer to the host computer allows further analysis such as the removal of plural scattering by spectral deconvolution. Elemental maps and line-scans can be recorded with real-time processing of energy loss data at each pixel. Images may be analyzed to provide quantitative information by means of pixel intensity histograms. If parameters for the background are stored at each pixel, the image data may sometimes be further processed to improve the signal-to-noise ratio.

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.

Aortic transmural serum protein transport: effect of concentration, time, and location.

The diffusive transport of certain 125I-labeled purified serum proteins, canine (C), human (H), and porcine (P) serum albumin (A), as well as human high-density lipoprotein (HDL), into the isolated deendothelialized intimal-medial thoracic aortic preparation was measured as a function of protein concentration (c0), intimal surface exposure time (T), and location (z) along the vessel at 21 degrees C. Selected proteins were studied in each of 11 canine preparations and 1 porcine preparation. The resulting uptake (M, nmol/cm2) was measured by direct gamma counting of specially excised fixed tissue specimens, and the transmural concentration distributions [c(xi), nmol/cm3] were calculated from electron probe X-ray microanalysis of the silver distributions across specially prepared microautoradiographs. The results showed 1) that the processes associated with the diffusive transport of CA, HA, PA, and HDL into the intimal-medial system are independent of c0, i.e., uptake is proportional to c0, 2) that the uptake of CA for short times appeared to be linear with T 1/2, 3) that the apparent wall-plasma partition coefficient for albumin rangers between 0.1 and 0.2, 4) that the apparent tissue albumin diffusion coefficient is approximately 2.7 X 10(-8) cm2/s, 5) that the transport processes for HA and CA are indistinguishable, 6) that the processes for the transport of HDL are two times slower than those for HA or CA, and 7) that the transport rate for albumin tends to decrease with z.

Quantitative microautoradiography of arteries: comparison of radioactivity to silver.

The local concentration of silver in developed aortic transmural microautoradiographs was compared to the corresponding 125I-labeled albumin radioactivity concentration [ci(x)] in the subjacent tissue. Silver [s(x)] was measured by electron probe X-ray microanalysis (EPA) and the corresponding ci(x) by direct gamma-ray counting. The results show 1) that the relationship between volume-averaged values of radioactivity (ci) and EPA signal (s) is adequately described by ci = Ks, where K is a proportionality constant, and 2) that ci(x) measured by EPA [i.e., Ks(x)] agrees closely with ci(s) measured direcly from en face microtomy slices of corresponding unfixed tissue specimens.