Energy-filtering transmission electron microscopy (EFTEM) in the elemental analysis of pseudoexfoliative material

Purpose. To obtain more information on the basic nature of the pathological matrix product accumulating in pseudoexfoliation (PEX) syndrome by analyzing its elemental composition at the subcellular level. Methods. Energy-filtering transmission electron microscopy (EFTEM), combining the two microanalytical techniques of electron spectroscopic imaging (ESI) and energy-loss spectroscopy (EELS), were performed on ultrathin sections of lens specimens with PEX syndrome using a transmission electron microscope equipped with an integrated electron energy filter. EFTEM is based on inner shell ionization of elements present in the sample giving rise to characteristic signals in well-defined energy-loss regions. The EEL-spectra, demonstrating the presence of a particular element by its specific electron energy-loss edge, were recorded with an integrated scintillator-photomultiplier-system. ESI generated graphic images of elemental localization in the sections after a process of background correction with an IBAS image analysis program. Energy-dispersive X-ray (EDX) analysis of PEX deposits on hydrated lenses was conducted by variable pressure scanning electron microscopy. Results. The ESI element distribution images of both intracapsular and supracapsular PEX material displayed high signals for nitrogen, sulfur, calcium, chlorine, and zinc in clear association with the PEX fibrils. The corresponding EEL-spectra confirmed the data obtained by ESI and showed the presence of the element-specific energy-loss edges. The presence of these elements in PEX fibrils was further confirmed by EDX analysis. No specific signals were obtained for phosphorus, oxygen, or aluminum. Conclusions. This study demonstrates the presence of nitrogen, sulfur, chlorine, zinc, and calcium both in mature and in aggregating PEX fibrils of the lens capsule. EFTEM proved to be a highly sensitive method for the microanalytical study of biological material with unknown composition, such as PEX material, at the subcellular level.