Use of a mass-thickness marker to estimate systematic errors and statistical noise in the detection of phosphorus by electron spectroscopic imaging

The element signal obtained from electron-energy-filtered micrographs depends on the systematic error in calculating the background and on the noise in the background-corrected image. Both systematic error and statistical fluctuation of the background can be assessed experimentally with a specimen that combines the element-containing feature with a mass-thickness marker. The approach is described for the mapping of phosphorus in turnip yellow mosaic viruses prepared on a supporting carbon film of variable thickness. The thickness modulations are produced by the additional deposition of heat-evaporated carbon through a second grid used as a mask. The three-window power-law method and the two-window difference method are compared. With the three-window power-law method, the mass-thickness modulations of the marker are still visible in the map, indicating a systematic error for the calculated background. In addition, the intensity profile over the area of the thick carbon film is broader than in the map corrected by the two-window method, indicating a higher level of noise. With the two-window difference method, mass-thickness contrast was practically eliminated due to an improved protocol that uses the mass-thickness marker to calculate the scaling factor: instead of scaling the grey-level of a single background feature, the pre-edge image is scaled to the contrast of the marker area in the image acquired at the element-specific energy loss.