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 in the electron microscope. However, the small size of the labels makes them very difficult to detect by conventional bright-field electron tomography. We have developed a technique based on quantitative scanning transmission electron microscopy (STEM) at a beam voltage of 300 kV and have demonstrated that it is possible to detect 11-gold atom clusters (undecagold) and 1.4 nm-diameter nanoparticles (nanogold). Initially, we simulated STEM images of gold clusters embedded in carbon using the NIST elastic scattering cross-section database. The simulated 2-D images of undecagold clusters embedded in a homogeneous matrix indicate that the cluster visibility is maximized for low inner collection semi-angles of the STEM annular dark-field detector (15-20 mrad). Our calculations also show that the visibility of undecagold clusters in 3D reconstructions is significantly higher than in 2D images when the clusters are embedded in an inhomogeneous matrix corresponding to local fluctuations in composition. Our experimental measurements show 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.
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