Electron energy loss spectroscopy microanalysis and imaging in the transmission electron microscope: example of biological applications

This paper reports original results obtained in our laboratory over the past few years in the application of both electron energy loss spectroscopy (EELS) and electron spectroscopy imaging (ESI) to biological samples, performed in two transmission electron microscopes (TEM) equipped with high-resolution electron filters and spectrometers: a Gatan model 607 single magnetic sector double focusing EEL serial spectrometer attached to a Philips 430 TEM and a Zeiss EM902 Energy Filtering TEM. The primary interest was on the possibility offered by the combined application of these spectroscopic techniques with those offered by the TEM. In particular, the electron beam focusing available in a TEM allowed us to perform EELS and ESI on very small sample volumes, where high-resolution imaging and electron diffraction techniques can provide important structural information. I show that ESI was able to improve TEM performance, due to the reduced chromatic aberration and the possibility of avoiding the sample staining procedure. Finally, the analysis of the oscillating extended energy loss fine structure (EXELFS) beyond the ionization edges characterizing the EELS spectra allowed me, in a manner very similar to the extended X-ray absorption fine structure (EXAFS) analysis of the X-ray absorption spectra, to obtain short-range structural information for such light elements of biological interest as O or Fe. The Philips EM430 (250-300 keV) TEM was used to perform EELS microanalysis on Ca, P, O, Fe, Al and Si. The assessment of the detection limits of this method was obtained working with well-characterized samples containing Ca and P, and mimicking the actual cellular matrix. I applied EELS microanalysis to Ca detection in bone tissue during the mineralization process and to P detection in the cellular membrane of erythrocytes treated with an anti-tumoral drug, demonstrating that the cellular membrane is a drug target. I applied EELS microanalysis and selected area electron diffraction (SAED) to study clay particle engulfed in the alveolar macrophages of human silicotic patients, demonstrating that the particle was surrounded by a Fe coating formed by ferritin. Moreover, the analysis of the EXELFS of Fe-L2,3 edge allowed me to study the mineral core of the ferritin, which showed great similarity to the hematite structure. Finally, I performed ESI in a Zeiss EM902 (80 keV) TEM to study the subcellular localization of the anti-neoplastic drug 4'-deoxy-4'-iododoxorubicin, analogous to adriamycin, in treated human breast tumoral cell, showing that this technique is useful in the identification of the drug target