Techniques for acquiring electron energy loss (EELS) maps in the BEIP field-emission STEM have been developed further. Computer-controlled compensation for specimen drift has been implemented to allow the long acquisition times (in excess of two hours) required to detect physiological concentrations of elements in cryosectioned tissue. This compensation has been achieved by periodically collecting fast elastic images during EELS mapping and using automated Fourier cross-correlation methods to determine shift vectors, which are then applied to correct for the drift. A new spectral acquisition mode has also been developed to reduce radiation damage in beam-sensitive biological specimens. After acquiring a fast digital elastic image, a binary mask of the area to be analyzed is generated and fed back into the digital scan generator. In this way, the EELS spectrum can be collected at low dose by distributing the incident electrons over any extended specimen area that has a complicated shape. In another development, EELS in combination with digital dark-field imaging has been used to determine the thicknesses of protein crystals, in order to perform more accurate three-dimensional electron crystallography so that data from different crystals can be successfully merged.
