Contrast Enhancement of the Brownish Horseradish Peroxidase-activated 3,3'-diaminobenzidine Tetrahydrochloride Reaction Product in Black and White Photomicrography by the Use of Interference Filters

An interference filter combination for contrast enhancement of the brownish, peroxidase-activated, 3,3'-diaminobenzidine tetrahydrochloride reaction product has been developed. This filter combination allows transmission of light in two narrow bands, representing those of the primary colors green (480-500 nm) and red (575-585 nm) as well as a broader band between 385-430 nm. The visual contrast enhancement of horseradish peroxidase-containing neurons caused by the interference filters was excellent. In particular, intracellular details were prominent in the presence of the interference filters compared with different blue absorption filters. The contrast enhancement was evaluated by microdensitometry of an Agfapan 25 emulsion onto which peroxidase-containing neurons in noncounterstained sections were exposed. The microdensitometry showed a neuron to background contrast enhancement of 30% compared to neutral gray filters and of 11% compared to a Kodak Wratten 80A blue absorption filter. The interference filters are available from the Laboratory of Technical Optics, the Technical University of Copenhagen, Building 307, Lundtoftevej 100, DK-2800 Lyngby, Denmark (order: DAB-interference filters, 30 mm). The interference filter principle can be used for contrast enhancement of other compound colors.