284 individual cells (DiGregorio et al. 1991). Other studies, using an end-point assay have also provided quantitative data for NBT reduction by activated leukocytes (e.g., Champelovier et al. 1993). A live cell imaging assay in which cytosolic Ca2+ was measured with the ﬂuorescence probe Fura-2 and O2- production was detected with NBT, was developed. When neutrophils were stimulated with a chemotactic peptide, localized production of O2- corre- lated with the location of a cytosolic ‘‘calcium cloud’’ (Davies et al. 1991). We have shown that the diformazan deposits in activated neutrophils can be monitored by laser scanning confocal microscopy using the reﬂectance Histochem Cell Biol (2008) 130:281–297 mode (Robinson and Batten 1989; Robinson and Badwey 1995) (see Figs. 2, 3). H2O2 detection in phagosomes with diaminobenzidine The presence of H2O2 within neutrophil phagosomes was initially inferred from the cytochemical experiments of Briggs et al. (1975a). Myeloperoxidase (MPO), present in azurophil granules of neutrophils, is delivered to the phagosome by fusion of azurophil granules with phago- somes. Myeloperoxidase utilizes H2O2 in the generation of hypochlorous acid, a potent microbicidal agent, in the Fig. 2 Cytochemical detection of sites of stimulated O2- generation with the NBT reaction and laser scanning confocal microscopy. A single neutrophil was allowed to adhere to a glass cover slip and was subsequently stimulated with 4 b-phorbol 12-myristate acetate in the presence of NBT is shown. The cell was then optically sectioned and viewed by transmitted light (a–f) and by confocal laser scanning microscopy in the reﬂectance mode (a0–f0). The non-confocal transmitted light images go from the substratum region (a) toward the apical portion of the cell (f). The reﬂectance images represent confocal sections corresponding to these different focal planes in the transmitted light images. Reaction product is present over the entire cell: however, there is heterogeneity in its distribution. Bar: 10 lm. Reprinted from Robinson and Badwey 1995 123