A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed Nanogold-labeled molecules

Abstract

We have developed methods to locate individual ligands that can be used for electron microscopy studies of dynamic events during endocytosis and subsequent intracellular trafficking. The methods are based on enlargement of 1.4 nm Nanogold attached to an endocytosed ligand. Nanogold, a small label that does not induce misdirection of ligand-receptor complexes, is ideal for labeling ligands endocytosed by live cells, but is too small to be routinely located in cells by electron microscopy. Traditional pre-embedding enhancement protocols to enlarge Nanogold are not compatible with high pressure freezing/freeze substitution fixation (HPF/FSF), the most accurate method to preserve ultrastructure and dynamic events during trafficking. We have developed an improved enhancement procedure for chemically-fixed samples that reduced autonucleation, and a new pre-embedding goldenlarging technique for HPF/FSF samples that preserved contrast and ultrastructure and can be used for high-resolution tomography. We evaluated our methods using labeled Fc as a ligand for the neonatal Fc receptor. Attachment of Nanogold to Fc did not interfere with receptor binding or uptake, and gold-labeled Fc could be specifically enlarged to allow identification in 2D projections and in tomograms. These methods should be broadly applicable to many endocytosis and transcytosis studies.

Biomolecular Imaging at High Spatial and Temporal Resolution In Vitro and In Vivo

“…However, recently C s -corrected microscopes have become available [35,36]. These have been developed by the materials-science community as an adjunct to image processing and are becoming more popular among the biological sciences [37]. Indeed, TEMs with both chromatic (C c ) and spherical (C s ) abberation correctors are now available with a sub-Ångstrom information limit [38].…”

“…An energy filter to remove inelastically scattered electrons would increase resolution further. For biological specimens, the EM also needs low dose imaging capabilities and a cryogenically-cooled specimen holder [37].…”

“…Commercial gold enhancement kits are available but in this case gave very high background. For this reason a modified protocol based on that developed by Morphew et al was used [37], which slowed down development time and decreased background labelling. Figure 5.13 clearly shows a non-transfected cell to the left of a transfected cell.…”

What transmission electron microscopes can visualize now and in the future

“…This technique has been developed in several laboratories and generally involves the substitution of water by organic solvents for plastic embedding at low temperature (Hess, 2007;McDonald, 2007). Although the method has been used very successfully (Buser et al, 2007;Giddings et al, 2001;He et al, 2007;Robertson, 2007;Segui-Simarro and Staehelin, 2006), the substitution step may induce artifacts also encountered by classical block-embedding techniques. The ultimate approach, again developed by Dubochet and co-workers, is the preparation of thin sections from vitrified ice blocks (Al-Amoudi et al, 2005, 2004aBouchet-marquis et al, 2007).…”

Low Temperature Specimen Preparation for Electron Tomography

“…Immuno-localization of antigens on the surface of sections prepared by RS/FSF is a useful method for ET, especially when serial tomograms are combined [2]. In addition, freeze-substitution based silver enhancement of Nanogold is useful for ET, particularly in cases where Nanogold is attached to endocytosed ligand which can be located during intracellular trafficking [3,4].…”