Quantitative analysis of electron energy‐loss spectra from ultrathin‐sectioned biological material

Abstract

SUMMARY A computer program for quantitative spectral analysis is proposed for the elemental analysis of biological material by electron energy‐loss spectroscopy in a conventional transmission electron microscope, the Zeiss EM902. Bio‐standards are used to test the performance of this program. The application of a simplex optimization method for curve‐fitting is proposed to separate the ionization edge from the background. Making use of Ce‐, Ca‐ and Fe‐bio‐standards, this method is compared with Egerton's well‐known two‐area method.

Direct visualization of intracellular calcium in rat osteoblasts by energy-filtering transmission electron microscopy

“…In biological samples, the calcium spectrum is superimposed on a rapidly falling background of random energy loss, and artifacts may result from scattering effects. The background is therefore subtracted from the experimental spectrum by computer-assisted processing (Ahn and Krivanek 1983;Sorber et al 1991;Reimer et al 1992) to obtain the net calcium-specific spectrum. The methods used for EELS and ESI acquisitions and background calculation have been described previously (Bordat et al 1998).…”

“…For energy losses above 100 eV, the background was assumed to follow an inverse power law I(E)=AE r , where I(E) is the intensity at energy loss E (De Buijn et al 1993). The pre-edge window (12-16 eV) used to calculate A and r by the Simplex optimization (Sorber et al 1991) for each spectrum was determined experimentally, and the calculated background was subtracted from the experimental spectrum. The relative section thickness t/l [t/ l=ln(I t /I o )], where l is a function of the specimen composition, was evaluated by the magnitude of the zero-loss peak I o (adjusted to 10 7 relative intensity) with respect to the intensity I t =0-100 eV (Reimer et al 1992).…”

Distribution of iron oxide nanoparticles in rat lymph nodes studied using electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI)

“…The net iron spectrum was obtained by subtracting the background. This is composed of random energy losses and was calculated by simplex optimization (18).…”

Comparison of the simplex method with several other methods for background-fitting for electron energy-loss spectral quantification of biological materials

“…Application of a three dimensional Simplex method (see Fig. 7) allowed us to judge the minimal number of channels in the r-range that was needed to get constant values for A, and r (for Caand Fe-Bio-standards see [7,8] …”

“…Th acquire good-quality reproducible spectra, we recently advocated the use of ultrathin sectioned Bio-standard [6][7][8] figure 6c.…”