Heavy metal contrast enhancement for the selective detection of gold particles in electron microscopical sections using electron spectroscopic imaging

In ultrathin sections for electron microscopy the evaluation of images generated with immunocytochemical or in situ hybridization techniques can become difficult if the colloidal gold label is very small and the underlying structure shows a strong heavy metal contrast caused by conventional uranium/lead staining. Normally, the problem is overcome by analyzing specimens at higher magnifications or by enlarging the gold grains in a secondary process called silver enhancement. We present a method to visualize the small gold particles with electron spectroscopic imaging (ESI). Three energy-filtered images acquired at energy losses of 0, 45 and 120 eV are analyzed by digital image treatment so as to allow the optical separation of the Au-markers from the image background. This protocol can be adapted for applications related to the automated counting of the label.