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

Abstract

SUMMARY Electron energy‐loss spectroscopy (EELS) has been used to determine elemental concentrations in biological specimens, consisting of ultrathin‐sectioned cells and tissues. Chelex100‐based Ca‐ and Fe Bio‐standards are used for elemental quantification to establish iron and calcium concentrations. These Bio‐standards, as well as the biological materials, are treated in a standard EM procedure such that ‘known’ and ‘unknown’ sites are located in one cross‐section. Uncertainties and variabilities present in the equations for calculating the concentration in the ‘unknown’ site (determined by comparing simplex‐fitted EEL spectra from Bio‐standards with those from tissue) are outlined in two examples. Using an H+ Bio‐standard, the matrix composition of such biological cell material is analysed, leading to values which approach each other closely. Quantitative EELS, using Chelex100‐based Bio‐standards, is advocated.

Quantitative electron spectroscopic imaging in bio‐medicine: Methods for image acquisition, correction and analysis

“…Exactly the same procedure is followed for the Bio-standard data set and the average R-value was Rst = 44 x lo-' at 3000x magnification. Sorber et al (1991) determined a concentration value of N,, = 1.6 atoms Fe/nm3 for the Chelex standard. The measurement scale of the unknown is now calibrated with these values, based on the expression in Eq.…”

“…In addition to the sequence of energy-filtered images required for quantification, the protocol also includes the acquisition of images for reference to the cell structure and morphology (with an energy-loss of 250 eV), for the assessment of radiation damage and specimen drift, for noise measurement and for the correction of several imaging effects. To measure the elemental concentrations in biological sections we co-embed a known biostandard for reference (Sorber, 1991). The protocol also demands the acquisition of at least one set of images for the analysis of the standard.…”

“…Values for @,A) can be obtained explicitly by modelling (see e.g. Leapman et a]., 1980), and by experimental measurement (Auerhammer et al, 1989) or this factor can be eliminated through the use of standards (Sorber et al, 1991). The quantification procedure can also be applied to each pixel location in a sequence of electron energy-loss filtered images to obtain quantitative information in a spatial context.…”

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.…”