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

Many questions about the metabolism of specific elements in the human body might be answered if elemental concentrations could be measured in situ in cells. With electron energy‐loss spectroscopic imaging (ESI), concentrations can potentially be determined with high spatial resolution. The theory of the quantification procedure has already been derived. Many practical instrument‐related problems, however, have to be solved. In the current research an energy‐filtering TEM is used and the image‐acquisition chain is examined in detail. Quantification requires images to be recorded over a large dynamic range. To solve this problem, the use of optical attenuation filters has been introduced. The use of the combination of a scintillator screen and a TV‐camera as a detection system has consequences for the processing of the data. Corrections for the camera photometric sensitivity and, to some extent, for shading are necessary. Further consequences of such a detection system for the correction of the element a‐specific spectral background and element detection are discussed. The derived methodology is tested in several ways and finally applied for the quantitative analysis of iron in liver parenchymal cells of a porphyria cutanea tarda patient.