On the feasibility of visualizing ultrasmall gold labels in biological specimens by STEM tomography

Abstract

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 300 kV for detecting 11-gold atom clusters (Undecagold) and 1.4 nm-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 (15-20 mrad). 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 freezesubstituted 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.

Combining high‐pressure freezing with pre‐embedding immunogold electron microscopy and tomography

“…NANOGOLD‐conjugates are ultra‐small markers with a gold core of just ≈1.4 nm, usually not recognizable in the grainy, noisy environment of cells processed for EM (except with certain high‐end EM‐imaging approaches; see, eg, Ref. ). Thus, the size of these particles must be increased by depositing metallic silver on the gold surface.…”

Liquid Scanning Transmission Electron Microscopy: Imaging Protein Complexes in their Native Environment in Whole Eukaryotic Cells

“…b: The cell is maintained in a saturated water vapor atmosphere, while a thin layer of water covers the cell for Liquid STEM using environmental scanning electron microscopy. in liquid , or in thick (biological) materials in general (Sousa et al, 2007). Note that inelastic scattering, typically occurring at smaller angles, and multiple scattering can be neglected .…”

Whole-Cell Analysis of Low-Density Lipoprotein Uptake by Macrophages Using STEM Tomography

“…STEM was used to acquire tilt series [14] of the whole-mount cells. The annular dark field (ADF) detector of STEM generates a much stronger contrast on gold than on cellular content such that gold is visible even in micrometers-thick cellular domains [15], [16]. The images mainly highlight the locations of the nanoparticles.…”