Limitations of beam damage in electron spectroscopic tomography of embedded cells.

Elemental mapping in the energy filtering transmission electron microscope (EFTEM) can be extended into three dimensions (3D) by acquiring a series of two-dimensional (2D) core-edge images from a specimen oriented over a range of tilt angles, and then reconstructing the volume using tomographic methods. EFTEM has been applied to imaging the distribution of biological molecules in 2D, e.g. nucleic acid and protein, in sections of plastic-embedded cells, but no systematic study has been undertaken to assess the extent to which beam damage limits the available information in 3D. To address this question, 2D elemental maps of phosphorus and nitrogen were acquired from unstained sections of plastic-embedded isolated mouse thymocytes. The variation in elemental composition, residual specimen mass and changes in the specimen morphology were measured as a function of electron dose. Whereas 40% of the total specimen mass was lost at doses above 10(6) e(-)/nm(2), no significant loss of phosphorus or nitrogen was observed for doses as high as 10(8) e(-)/nm(2). The oxygen content decreased from 25 + or - 2 to 9 + or - 2 atomic percent at an electron dose of 10(4) e(-)/nm(2), which accounted for a major component of the total mass loss. The specimen thickness decreased by 50% after a dose of 10(8) e(-)/nm(2), and a lateral shrinkage of 9.5 + or - 2.0% occurred from 2 x 10(4) to 10(8) e(-)/nm(2). At doses above 10(7) e(-)/nm(2), damage could be observed in the bright field as well in the core edge images, which is attributed to further loss of oxygen and carbon atoms. Despite these artefacts, electron tomograms obtained from high-pressure frozen and freeze-substituted sections of C. elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins.