Action of gold chloride ("gold toning") on silver-enhanced 1 nm gold markers.

In conventional immunoelectron microscopy (IEM), very small colloidal gold particles (0.8-3 nm), or the gold compound Nanogold (1.4 nm) are silver-enhanced for easy detection. However, silver enhancement has drawbacks. First, the silver layer is dissolved during fixation with osmium tetroxide, even if the concentration and incubation time are strongly reduced during pre-embedding labeling experiments in transmission electron microscopic (TEM) and scanning electron microscopic (SEM) studies. Second, after exposure to the electron beam the silver layer may migrate on the section or the whole particles may disappear. Sometimes silver migration can be observed even without irradiation. This effect strongly hampers reinvestigation of previously inspected areas, after some time of storage. In both cases, gold chloride treatment after silver enhancement is sufficient to completely protect the silver-enhanced 1 nm gold markers. Gold chloride treatment is part of the so-called "gold toning" procedure, which is a method used to substitute and/or cover the silver by a layer of gold. It can be applied in TEM and SEM experiments. As a serious drawback, gold chloride treatment slightly reduces the size of both unenhanced and silver-enhanced gold particles and can lead to disintegrated silver/gold particles. Therefore, this technique is useful for pre-embedding IEM, on-(resin)section, and ultrathin cryosection labeling experiments. However, it appears to be unsuitable for double-labeling studies using different gold sizes, for quantitation experiments, and in SEM.