Comparison of Techniques for EELS Mapping in Biology

Elemental mapping of biological specimens by electron energy loss spectroscopy (EELS) can be carried out both in the scanning transmission electron microscope (STEM), and in the energy-filtering transmission electron microscope (EFTEM). Choosing between these two approaches is complicated by the variety of specimens that are encountered (e.g., cells or macromolecules; cryosections, plastic sections or thin films) and by the range of elemental concentrations that occur (from a few percent down to a few parts per million). Our aim here is to consider the strengths of each technique for determining elemental distributions in these different types of specimen.On one hand, it is desirable to collect a parallel EELS spectrum at each point in the specimen using the ‘spectrum-imaging’ technique in the STEM. This minimizes the electron dose and retains as much quantitative information as possible about the inelastic scattering processes in the specimen. On the other hand, collection times in the STEM are often limited by the detector read-out and by available probe current. For example, a 256 x 256 pixel image in the STEM takes at least 30 minutes to acquire with read-out time of 25 ms. The EFTEM is able to collect parallel image data using slow-scan CCD array detectors from as many as 1024 x 1024 pixels with integration times of a few seconds. Furthermore, the EFTEM has an available beam current in the µA range compared with just a few nA in the STEM. Indeed, for some applications this can result in a factor of ~100 shorter acquisition time for the EFTEM relative to the STEM. However, the EFTEM provides much less spectral information, so that the technique of choice ultimately depends on requirements for processing the spectrum at each pixel (viz., isolated edges vs. overlapping edges, uniform thickness vs. non-uniform thickness, molar vs. millimolar concentrations).