286 Histochem Cell Biol (2008) 130:281–297 Fig. 4 Electron micrograph of a human neutrophil that has ingested E. coli minicells; the neutrophil was subsequently reacted for the cytochemical detection of MPO activity using the DAB method with exogenous H2O2. Black electron dense deposits are present in intracellular granules, the azurophil granules (asterisks), and within a population of phagosomes (arrows). Under these incubation conditions, there were some phagosomes that had not yet acquired MPO (double arrows). The cytochemical reaction for the detection of MPO activity following ingestion of minicells was carried out as we have described (Fox et al. 1987). Bar: 1 lm bright-ﬁeld optical microscopy. This problem was over- come as cerium perhydroxide could be converted to other products visible by bright-ﬁeld microscopy (Zimmerman and Halbhuber 1985; Okada et al. 1987; Angerm u¨ller and Fahimi 1988; Halbhuber et al. 1988). It was subsequently found that the cerium perhydroxide reaction product can be detected directly, without conversion to another product, using the reﬂectance mode of the laser scanning confocal microscope (Robinson and Batten 1990). This latter method allows for direct visualization of the cerium reac- tion product and permits precise localization by means of confocal optical sectioning and collection of three-dimen- sional data sets. Cerium has proven to be a versatile tool for enzyme cytochemistry since it can also be used in the detection of several phosphatases and has several advan- tages over older methods for detection of these enzymes (Robinson and Karnovsky 1983a, b). The use of cerium in oxidase and phosphatase enzyme cytochemistry has been reviewed (Van Noorden and Fredericks 1993; Halbhuber et al. 1994; Karnovsky 1994). 123 Fig. 5 Electron micrograph illustrating the cytochemical detection of H2O2 with the cerium method in a neutrophil that was stimulated by phagocytosis. This thin-section electron micrograph is of a cell that had phagocytized zymozan particles (yeast cell walls) and was then incubated for cytochemistry as described previously (Briggs et al. 1975b). Electron-dense reaction product is present on portions of the cell surface (arrowheads) and in some phagosomes. In this cell proﬁle, ﬁve zymozan particles appear to be within phagosomes (P1–5), while two additional particles are bound to the cell surface, but are incompletely internalized (P6–7). Note that some particles (P1–3) have reaction product surrounding the luminal aspect of the phago- somes (arrows), while other particles (P4–5) lack reaction product in the phagosomes (double arrows). Portions of the nucleus (N) are evident. These results indicate an asymmetry in the spatial and/or temporal delivery of H2O2 to phagosomes. Bar: 1 lm. Reprinted from Robinson and Badwey 1995 When neutrophils are incubated with phagocytizable particles, such as yeast cell walls, in the cerium-based cytochemical medium developed by Briggs et al. (1975b), the reaction product is evident within phagosomes and at the cell surface. These results were conﬁrmed in other studies (Badwey et al. 1980; Ohno et al. 1982). There are also deposits of reaction product in vesicle-like structures in the cytoplasm (see more below). We have noted that typically phagosomes can vary with regard to the apparent amount of reaction product per phagosome with some phagosomes lacking reaction product altogether suggesting an asymmetric delivery of H2O2 to phagosomes (Fig. 5). Similar asymmetry in the delivery of ROS to phagosomes was obtained in living cells by Soret band transmitted light microscopy of neutrophils that phagocytized antibody- coated red blood cells (Petty et al. 1992). Grifﬁths (2004) proposes that heterogeneity in phagosomes within the same