Dual-axis electron tomography of biological specimens: Extending the limits of specimen thickness with bright-field STEM imaging.
The absence of imaging lenses after the specimen in the scanning transmission electron microscope (STEM) enables electron tomography to be performed in the STEM mode on micrometer-thick plastic-embedded specimens without the deleterious effect of chromatic aberration, which limits spatial resolution and signal-to-noise ratio in conventional TEM. Using Monte Carlo calculations to simulate electron scattering from gold nanoparticles situated at the top and bottom surfaces of a plastic section, we assess the optimal acquisition strategy for axial bright-field STEM electron tomography at a beam-energy of 300keV. Dual tilt-axis STEM tomography with optimized axial bight-field detector geometry is demonstrated by application to micrometer-thick sections of beta cells from mouse pancreatic islet. The quality of the resulting three-dimensional reconstructions is comparable to that obtained from much thinner (0.3-micrometer) sections using conventional TEM tomography. The increased range of specimen thickness accessible to axial STEM tomography without the need for serial sectioning enables the 3-D visualization of more complex and larger subcellular structures.
Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA.
Alioscka A
AA
Sousa
Afrouz A
AA
Azari
Guofeng
G
Zhang
Richard D
RD
Leapman
D000818:Animals; D055032:Electron Microscope Tomography / Q000379:methods*; D050417:Insulin-Secreting Cells / Q000648:ultrastructure; D051379:Mice; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D009010:Monte Carlo Method