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

Purpose. To obtain more information on the basic nature of the pathological matrix product accumulating in pseudoexfoliation (PEX) syndrome by analyzi ng its elemental composition at the subcellular level. Methods. Energy-filtering transmission electron microscopy (EFTEM), combini ng the two microanalytical techniques of electron spectroscopic imaging (ES I) and energy-loss spectroscopy (EELS), were performed on ultrathin section s of lens specimens with PEX syndrome using a transmission electron microsc ope equipped with an integrated electron energy filter. EFTEM is based on i nner shell ionization of elements present in the sample giving rise to char acteristic signals in well-defined energy-loss regions. The EEL-spectra, de monstrating the presence of a particular element by its specific electron e nergy-loss edge, were recorded with an integrated scintillator-photomultipl ier-system. ESI generated graphic images of elemental localization in the s ections after a process of background correction with an IBAS image analysi s program. Energy-dispersive X-ray (EDX) analysis of PEX deposits on hydrat ed lenses was conducted by variable pressure scanning electron microscopy. Results. The ESI element distribution images of both intracapsular and supr acapsular PEX material displayed high signals for nitrogen, sulfur, calcium , chlorine, and zinc in clear association with the PEX fibrils. The corresp onding EEL-spectra confirmed the data obtained by ESI and showed the presen ce of the element-specific energy-loss edges. The presence of these element s 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, chlo rine, zinc, and calcium both in mature and in aggregating PEX fibrils of th e lens capsule. EFTEM proved to be a highly sensitive method for the microa nalytical study of biological material with unknown composition, such as PE X material, at the subcellular level.