Quantitative electron spectroscopic imaging in bio‐medicine: evaluation and application

Abstract

Electron spectroscopic imaging (ESI) with the energy-filtering transmission electron microscope enables the investigation of chemical elements in ultrathin biological sections. An analysis technique has been developed to calculate elemental maps and quantitative distributions from ESI sequences. Extensive experience has been obtained with a practical implementation of this technique. A procedure for more robust element detection has been investigated and optimized. With the use of Fe-loaded Chelex beads, the measurement system has been evaluated with respect to the linearity of the element concentration scale, the reproducibility of the measurements and the visual usage of image results. In liver specimens of a patient with an iron storage disease the detectability of iron was tested and we tried to characterize iron-containing components. The concentration measurement scale is approximately linear up to a relative section thickness of approximately equal to 0.5. Monitoring of this parameter is therefore considered to be important. The reproducibility was measured in an experiment with Fe-Chelex. The iron concentration differed by 6.4% between two serial measurements. Element distributions are in many applications interpreted visually. For this purpose the frequently used net-intensity distributions are regarded as unsuitable. For the quantification and visual interpretation of concentration differences mass thickness correction has to be performed. By contrast, for the detection of elements the signal-to-noise ratio is the appropriate criterion. Application of ESI analysis demonstrated the quantitative chemical capabilities of this technique in the investigation of iron storage diseases. Based on an assumed ferritin iron loading in vivo, different iron components can be discerned in liver parenchymal cells of an iron-overloaded patient.

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

“…Under optimal conditions the EELS system can detect about 2.2 mmol/kg dry weight Ca in a 730--thick biological specimen (Ho et al 1999). Although the absolute thickness of the sections was not available in our study, the relative thickness t/l, where l is a function of the specimen composition, especially of density (Yang and Egerton 1995;Shi et al 1996), was maintained below 0.5, the recommended limit for analytical purposes (Beckers et al 1996). Taking these precautions, scarce calcium signals were recorded from osteoblasts maintained in buffer, as reported by other investigators.…”

“…The threshold for ionomycin-treated samples (171€7 arbitrary units) was the same as that for the controls (174€6 arbitrary units). The calcium distribution (in red) was superimposed onto the images obtained below the carbon-K edge (250 eV; inversed contrast; Beckers et al 1996). The number of confluent red pixels/mitochondrion (10-16 contiguous pixels) was counted on enlarged images (12,000) on a selected number of osteoblasts: 46 ex vivo ionomycin-treated osteoblasts (17 controls) and 38 cultured ionomycin-treated osteoblasts (27 controls).…”

Calcium distribution in high-pressure frozen bone cells by electron energy loss spectroscopy and electron spectroscopic imaging

“…λ is the total inelastic mean free path and depends from the specimen composition (Yang and Egerton 1995;Beckers et al 1996).…”

“…Semiquantitative data were obtained according to the procedure described by De Bruijn et al (1993) and Beckers et al (1996). After subtraction of the continuum component, the net edge intensity under the Ca-L 2,3 edge, integrated over a predefined energy-loss region, was related to the total transmitted intensity.…”

Comment to “Mapping and Characterization of Iron Compounds in Alzheimer's Tissue”

“…Elemental maps of iron in ferritin have been obtained by EELS since 1982 [7] because it is an ideal standard ATEM test specimen [6,8]. EELS has been used for the analysis of iron and iron/ferritin in several human pathologies (see [9][10][11][12] for example). Recently, EELS has been combined with electron tomography to study the 3D distribution of elements in biological samples [13], particularly iron in degenerating neurons in mice with abnormal regulation of iron metabolism [14].…”