Reprint of "Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)" [J. Struct. Biol. 160 (2007) 35-48].

We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides 3-D distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2-D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L(2,3) edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell's nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7 nm-sized voxel.