Localization of chromophore absorption signals in TEM with an improved prism-mirror-prism filter

A corrected prism-mirror-prism electron energy filler with curved entrance and exit faces was designed and incorporated into a Zeiss EM902 transmissio n electron microscope. The installation of the new filter required little m odification to the existing microscope geometry and electronics. The filter had an energy resolution of 1.1 eV over the full image plane (acceptance h alf angle 10 mradian). The improved energy resolution was applied in studie s of the low electron energy loss region that includes molecular orbital ex citations or chromophore energy absorptions. Chromophore signal behaviour u nder electron irradiation was characterized using embedded crystals of hema tin and of the dye mercury orange. Images of these crystals confirmed the e xpected decrease in signal intensity on shifting the selected energy loss f rom the region of molecular orbital excitations (less than similar to 5 eV) to higher energy losses. Electron irradiation-induced fading of the chromo phore signal from hematin and mercury orange yielded similar lie dose value s of 1.1x10(5) e(-) nm(-2) and 1.4x10(5) e(-) nm(-2) respectively. In a cel lular context, chromophore signals were obtained from energy-filtered image s of RIF-1 cell sections containing the photosensitizer chlorin e(6) and fr om sections of BS-C-1 cells with cytoskeletal labelling via FITC-conjugated antibodies. The respective signals were extracted using a dose-dependent m ethod or a shift in selected energy. Chromophore signal distributions were in agreement with fluorescence light microscopic images, but provided detai l at higher spatial resolution.