Engery-filtering transmission electron microscopy of biological specimens.

By energy-filtering transmission electron microscopy (EFTEM) electrons can be separated by their energy losses. An electron-energy filter, added to the microscope column allows the measurement of the energy distribution of transmitted electrons that have lost energy (< 2,000 eV, with an energy resolution of approximately 1 eV). These filtered electrons, recorded either as a spectrum or as an image, are composed of two parts superimposed on top of each other: (a) the unspecific energy-loss population (= the continuum) and (b) the specific element-related energy-loss population (= the edges). At the edges, electron data in spectra and images are mathematically processed, to obtain the desired element-related net-intensity values or images. These data are related to the total transmitted electron intensity, from the zero- and low-loss spectral region giving the relative spectralor image intensity rations ((S)R*x, (I)R*x), which can be related to the element concentration. The acquisition of the zero-loss and low-loss data is hampered by the restricted dynamic range of the TV camera. By improvements through the introduction of calibrated attenuation filters in the optical path to the TV-camera, more reliable values for (S)R*x and (I)R*x can be acquired. By addition of Bio-standards adjacent to the tissue, a "known" and "unknown" concentration of the element present in the same ultrathin section and the "bias" in the concentration estimation, can be obtained. Some practical examples are given for the estimation of the iron cencentration in siderosomes, boron in melasosomes and calcium in calcium oxalate monohydrate crystals.