Histochem Cell Biol (2008) 130:281–297 and release of superoxide from neutrophils in response to different stimulatory agents (Fig. 1) (Badwey et al. 1980). The rate of O2- released from neutrophils in response to a variety of different agonists has been summarized (Robin- son and Badwey 1995). An overview of the various methods used to measure reactive oxygen species and limitations associated with these methods has been presented (Tarpey et al. 2004). There are several excellent reviews describing the components of the NADPH oxidase and the assembly of these components into the multiprotein complex capable of producing superoxide (Lambeth 2000; Babior et al. 2002; Vignais, 2002; Bokoch and Knaus 2003; Roos et al. 2003; Babior 2004; Cross and Segal 2004; Nauseef 2004; Groemping and Rittinger 2005; Matute et al. 2005; Sheppard et al. 2005; Dagher and Pick 2007; Nauseef 2007). Therefore a detailed analysis of this system will not be presented herein; instead, a distillation of the major features of the oxidase will be given. As is the case for most aspects of cell function, location is a key factor. This is certainly the case for the NADPH oxidase. Indeed, separation of different components of the oxidase in speciﬁc locations within the cell account for its dormancy in unstimulated cells. This is important since products of the oxidase are highly reactive and can lead to cell and tissue damage. Thus activation of the oxidase should occur only when necessary. It is only after appropriate stimulation that all of the components of the oxidase come together in a coordinated manner that this complex multicomponent molecular system is functional in intact cells (see below). The so-called cell-free system in which homogenized neutrophils were subjected to ultracentrifugation to gene- rate cytosol and membrane fractions have been important in sorting out the various components of the NADPH oxidase (Bromberg and Pick 1984; Heyneman and Ver- cauteren 1984; Curnutte 1985; McPhail et al. 1985). With this approach in hand, investigators could take membranes from one source and cytosol from another source and mix them together and assay for O2- production [reviewed in Dagher and Pick 2007]. For example, membranes from normal neutrophils and cytosol from neutrophils from patients with chronic granulomatous disease (CGD) could be mixed, and vice versa. In addition to this genetic approach, recombinant forms of components of the oxidase complex, mutated forms of these proteins, chimeric pro- teins, and synthetic peptides have also been used in the cell-free system to analyze the NADPH oxidase (Dagher and Pick 2007). A large number of experiments have been carried out by several groups using a variety of approaches to study the NADPH oxidase in cell-free preparations. Taken together, these data have been crucial in leading to our current understanding of the components, their 283 assembly, and regulation of the phagocyte NADPH oxidase. The current model for the NADPH oxidase has the catalytic component, ﬂavocytochrome b588, located in the membrane. This heterodimeric ﬂavocytochrome is com- posed of the proteins gp91phox and p22phox (Segal and Jones 1978; Dinauer et al. 1987; Parkos et al. 1988). The other components of the oxidase complex are in the cytoplasm. It is only after cell stimulation that the cytoplasmic compo- nents migrate to the membrane and associate with the gp91phox and p22phox complex. The cytosolic components of the oxidase consist of the proteins p67phox, p47phox, and p40phox, which appear to exist as a complex awaiting mobilization to join the cytochrome b558 upon cell stimu- lation (Wientjes et al. 1993, 1996; Zhan et al. 1998; Lapouge et al. 2002). An additional component of the NADPH oxidase is the GTP-bound form of Rac (Quinn et al. 1993; Abo et al. 1994; Bokoch and Diebold 2002). Until cell activation, Rac exists in association with RhoGDI in the GDP-bound form; upon cell stimulation, it dissociates from RhoGDI, exchanges GDP for GTP, and binds the NADPH oxidase (Bokoch et al. 1994; Kreck et al. 1996). As noted earlier, there are several excellent reviews covering the composition and assembly of phagocyte NADPH oxidase that have appeared since the turn of the century and the interested reader is referred to those pub- lications. The present review will focus on the role microscopy has played in our understanding of the phagocyte NADPH oxidase. Microscopy in the study of the respiratory burst The tetrazolium method: detection of superoxide Early microscopy studies of the respiratory burst in neu- trophils relied on the dye nitroblue tetrazolium (NBT). Indeed, NBT was used to diagnose the absence of the respiratory burst in neutrophils from CGD patients (Nathan et al. 1969). The so-called NBT test predated the discovery that CGD neutrophils were unable to generate O2- (Curnutte et al. 1974). The chemical basis of the NBT test has been presented elsewhere and will not be dis- cussed herein (Auclair and Voisin 1985). The NBT reaction was adapted for electron microscopy but it was noted that the diformazan reaction product lacked sufﬁ- cient electron density to be useful in this application (Briggs et al. 1975a). The tetrazolium 3-[4,5-dimethyl- thiazol-2-yl]-2,5-diphenyltetrazolium has also been used to monitor the respiratory burst (Pruett and Loftis 1990; Oez et al. 1993). A quantitative video-microscopy assay using NBT was used to show heterogeneity in O2- production among 123