Reprint of "On the feasibility of visualizing ultrasmall gold labels in biological specimens by STEM tomography" [J. Struct. Biol. 159 (2007) 507-522].
Labeling with heavy atom clusters attached to antibody fragments is an attractive technique for determining the 3D distribution of specific proteins in cells using electron tomography. However, the small size of the labels makes them very difficult to detect by conventional bright-field electron tomography. Here, we evaluate quantitative scanning transmission electron microscopy (STEM) at a beam voltage of 300kV for detecting 11-gold atom clusters (Undecagold) and 1.4nm-diameter nanoparticles (Nanogold) for a variety of specimens and imaging conditions. STEM images as well as tomographic tilt series are simulated by means of the NIST Elastic-Scattering Cross-Section Database for gold clusters embedded in carbon. The simulations indicate that the visibility in 2D of Undecagold clusters in a homogeneous matrix is maximized for low inner collection semi-angles of the STEM annular dark-field detector (15-20mrad). Furthermore, our calculations show that the visibility of Undecagold in 3D reconstructions is significantly higher than in 2D images for an inhomogeneous matrix corresponding to fluctuations in local density. The measurements demonstrate that it is possible to detect Nanogold particles in plastic sections of tissue freeze-substituted in the presence of osmium. STEM tomography has the potential to localize specific proteins in permeabilized cells using antibody fragments tagged with small heavy atom clusters. Our quantitative analysis provides a framework for determining the detection limits and optimal experimental conditions for localizing these small clusters.
Sousa
A A
AA
National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Building 13, Room 3N17, 13 South Drive, Bethesda, MD 20892-5766, USA.
Aronova
M A
MA
Kim
Y C
YC
Dorward
L M
LM
Zhang
G
G
Leapman
R D
RD