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 degrees at 5 degrees 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 10(7)e/nm2 was required to record two-axis tilt series, specimens were sufficiently stable to allow image alignment and tomographic reconstruction.
Leapman
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RD
Division of Bioengineering and Physical Science, ORS, National Institutes of Health, Building 13, 3N17, Bethesda, MD 20892, USA. leapman@helix.nih.gov
Kocsis
E
E
Zhang
G
G
Talbot
T L
TL
Laquerriere
P
P
D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry*; D005615:Freezing; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods; D010758:Phosphorus / Q000032:analysis*; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D014057:Tomography, X-Ray Computed
D010758:Phosphorus