Molecular mass determination by STEM and EFTEM: a critical comparison

The concept of molecular mass determination by transmission electron micros copy (TEM) was first presented in 1962 by Zeitler and Bahr. It is based on the approximately linear relationship between the mass thickness of a speci men and the fraction of beam electrons scattered by it. This single-particl e-type mass measuring method complements bulk-type mass determination metho ds such as, for example, ultracentrifugation, mass spectrometry, gel electr ophoresis or column chromatography. With only nano- to micromolar amounts o f sample required, not only the mass of a biomolecule can be determined, bu t also the mass-per-length or the mass-per-area of supramolecular assemblie s such as filaments or sheets can readily be evaluated. As mass determinati on by TEM is an imaging method and hence enables single-particle evaluation , a visual assessment of the sample is obtained, in terms of particle morph ologies that fall into a particular mass range. The development of the scan ning transmission electron microscope (STEM) together with the introduction of fast, high-sensitivity electron detectors and image processing computer s in the seventies improved the accuracy and ease of TEM-based mass determi nation considerably. Moreover, progress in the development of energy-filter ing transmission electron microscopes (EFTEM) and the availability of scien tific-grade slow-scan CCD cameras has now opened mass determination by elec tron scattering at the single-particle level to a wider circle of users wit hout the need of customizing the instrument. Comparative experiments assist ed by Monte Carlo simulations documented that for all practical purposes, t he accuracy of mass determination by EFTEM is the same as that by STEM. Thi s investigation also focused on radiation damage which is one factor affect ing the accuracy of mass determination of biological materials, together wi th instrumental limitations and specimen preparation. The effect of beam-in duced mass loss can be reduced by similar to 30% if the energy of the incid ent electron beam is increased from 80 to 120 keV. Moreover, further improv ement of the radiation resistency by a factor of similar to 7 is achieved b y cooling the specimen to -130 degrees C. (C) 1999 Elsevier Science Ltd. Al l rights reserved.