Three-dimensional distributions of elements in biological samples by energy-filtered electron tomography

By combining electron tomography with energy-filtered electron microscopy, we have shown the feasibility of determining the three-dimensional distributions of phosphorus in biological specimens. Thin sections of the nematode, Caenorhabditis elegans were prepared by high-pressure freezing, freeze-substitution and plastic embedding. Images were recorded at energy losses above and below the phosphorus L2,3 edge using a post-column imaging filter operating at a beam energy of 120 keV. The unstained specimens exhibited minimal contrast in bright-field images. After it was determined that the specimen was sufficiently thin to allow two-window ratio imaging of phosphorus, pairs of pre-edge and post-edge images were acquired in series over a tilt range of ±55° at 5° increments for two orthogonal tilt axes. The projected phosphorus distributions were aligned using the pre-edge images that contained inelastic contrast from colloidal gold particles deposited on the specimen surface. A reconstruction and surface rendering of the phosphorus distribution clearly revealed features 15–20 nm in diameter, which were identified as ribosomes distributed along the stacked membranes of endoplasmic reticulum and in the cytoplasm. The sensitivity of the technique was estimated at <35 phosphorus atoms per voxel based on the known total ribosomal phosphorus content of approximately 7000 atoms. Although a high electron dose of approximately 107 e/nm2 was required to record two-axis tilt series, specimens were sufficiently stable to allow image alignment and tomographic reconstruction.