Energy-Filtered Electron Tomography

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We have developed a technique called quantitative electron spectroscopic tomography (QuEST) for imaging the three-dimensional distribution of specific chemical elements in cells. A 300 kV field-emission transmission electron microscope (TEM) equipped with an advanced imaging filter is used to collect a series of 2-D elemental maps for a range of specimen tilt angles. Acquisition is controlled by means of flexible computer scripts, which enable correction for specimen drift and defocus between successive tilt angles. Projected 2-D elemental distributions are obtained by acquiring images above and below characteristic core-edges in the energy-loss spectrum and by subtracting the extrapolated background intensity at each pixel. We have implemented a dual-axis simultaneous iterative reconstruction technique (SIRT) to reconstruct the 3-D elemental distribution. By applying a thickness correction algorithm that takes into account plural inelastic scattering, and by incorporating scattering cross sections for excitation of core-shell electrons, we have shown that it is possible to quantify the elemental distributions in terms of the number of atoms per voxel. By using correlative light microscopy and 3-D phosphorus imaging, experiments are in progress to map the distribution of DNA in specific domains of cell nuclei, where macromolecular complexes are involved in regulation of genes. Preliminary results have been obtained showing the feasibility of using a dual fluoro-nanogold labeled antibody to image specific proteins contained within the chromatin insulator body complex. The proteins can be tracked in the optical microscope using the fluorescence tag, after which the gold nanoparticle tags can be visalized in 3D using electron tomography in the scanning transmission electron microscope (STEM) mode. Then EFTEM tomography is used to determine the distribution of DNA in the vicinity of the insulator body complex. We have also developed methods for quantitative energy-filtered TEM imaging of other important biological elements like calcium, which requires even more precise correction for plural scattering than does phosphorus mapping. EFTEM imaging of calcium distributions is being applied to study the regulation of calcium in rapidly frozen and cryosectioned neurons. This approach is complementary to energy-dispersive x-ray spectroscopy and electron energy-loss spectroscopy menasurements using a focused-probe in the scanning transmission electron microscope (STEM). EFTEM enables much larger specimen areas to be analyzed but with lower sensitivity.