EXPERIMENTS WITH A HIGH RESOLUTION FIELD-EMISSION STEM

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Further experiments have been performed to assess new applications of parallel-detection electron energy loss spectroscopy in the field-- emission STEM. This technique provides a high sensitivity for microanalysis of certain important biological elements (such as calcium), the physiological concentrations of which are rather low in cells. Application of parallel-EELS mapping to the analysis of freeze-dried cryosections provides a means of detecting small amounts of calcium in structures with a diameter of approximately 50 nm. Detector pattern noise due to channel gain variations can be reduced by acquiring difference spectra at each pixel. By segmenting nitrogen maps that reflect the structure through the protein distribution, it is possible to sum spectra from specific compartments. It has been found that useful data can be collected at 100 keV beam energy from freeze-dried cryosections cut to a nominal thickness of 100 nm. The analysis results in a sensitivity of +/-0.4 mmol Ca/kg dry weight, with a total acquisition time of 400 seconds, a significant improvement over that achievable with energy-dispersive x-ray spectroscopy. Another application of EELS is the measurement of precise protein crystal thickness for application to electron crystallography. This approach has been tested on glucose-embedded crotoxin, and results show that the thickness determination can be performed to an accuracy of one-half a unit cell.