An inherent limitation to the immunogold technique is that the gold particle is around 15-30 nm away from the site to which the primary antibody is bound (when using a primary and secondary antibodies labeling strategy). The precise location of the targeted molecule can therefore not be accurately calculated. Gold particles can be created with a diameter of 1 nm (or lower) but another limitation is then realized—at these sizes the gold label becomes hard to distinguish from tissue structure.
Thin sections are required for immunogold labeling and these can produce misleading images; a thin slice of a cell component may not give an accurate view of its three-dimensional structure. For example, a microtubule may appear as a spike depending on which plane the sectioning occurred. To overcome this limitation serial sections can be taken, which can then be compiled into a three-dimensional image.
A further limitation is that antibodies and gold particles cannot penetrate the resin used to embed samples for imaging. Thus, only accessible molecules can be targeted and visualized. Labeling prior to embedding the sample can reduce the negative impact of this limitation.