292 photobleaching does not occur; and (3) generation of ROS may be dissected further with speciﬁc chemilumigenic probes. The above arguments notwithstanding, ﬂuorescence microscopy in its various forms has been very important in cell biological studies, including those with phagocytic leukocytes. This is particularly so for live-cell imaging of stimulated leukocytes. Imaging procedures to monitor the endogenous autoﬂuorescence signal from NAD(P)H during activation of the NADPH oxidase have been developed (Liang and Petty 1992). This procedure, when coupled with monitoring ROS generation by ﬂuorescence microscopy, has provided intriguing data on metabolic waves in these cells and was discussed in an earlier section of this review (Kindzelskii and Petty 2002). Other oscillations, ranging from structural to biochemi- cal, have been observed in neutrophils (reviewed in Petty 2001). Also as already noted, this same group used Soret band (absorption contrast) transmitted light to monitor changes in hemoglobin in phagocytosed erythrocytes (Petty et al. 1992). Using high-speed ﬂuorescence imaging (shutter speeds *100 ns), it was possible to make ‘‘stop action movies’’ of signaling events such as calcium waves in neutrophils that had phagocytosed (Petty 2006). In this case, a calcium wave moves around the periphery of the cell and then jumps to the phagosome when it moves nearby. Rapid image acquisition methodology enabled these observations and should continue to provide new insights into cellular behavior in future work. Other advanced imaging modalities have been employed for studying the phagocyte NADPH oxidase (reviewed in Van Manen et al. 2006). The distribution of cytochrome b558 has been imaged in resting and phagocytosing neu- trophils at the single cell level by confocal resonance Raman microscopy (van Manen et al. 2003). In this case, Raman spectroscopy was used to visualize the cytochrome b558 directly rather than with a labeling procedure as in immunocytochemistry. While the cytochrome b558 accu- mulates in the region of ingested particles, the spatial resolution was rather low and thus does not provide deﬁnitive localization. The authors noted that individual speciﬁc granules were not resolved using this imaging modality. Reﬁnements in this instrumentation may improve spatial resolution. The immunoﬂuorescence localization of gp91phox in hematopoietic stem cells mobilized by granu- locyte-colony stimulating factor was determined by near ﬁeld scanning optical microscopy (NSOM) (Frassanito et al. 2008). With NSOM imaging, they report the presence of ‘‘specialized clusters’’ and also that ‘‘protein domains’’ in the 300–600 nm size range could be detected. The precise nature of these small protein domains remains to be determined. 123 Histochem Cell Biol (2008) 130:281–297 The use of cell lines that display certain properties of primary neutrophils have advantages over freshly isolated neutrophils for some studies. The cultured cells (e.g., PLB- 985 and HL-60 cells) can be manipulated in ways that are not readily applicable to primary neutrophils. For example, they can be transfected to express components of the NADPH oxidase that are tagged with ﬂuorescent proteins such that these components can be monitored in living cells under various conditions with real-time ﬂuorescence microscopy. An analysis of this expanding area is beyond the scope of this review; however, some examples are cited to illustrate the potential for these types of experiments. Fluorescence fusion proteins have been used to study the interaction of p67phox and Rac2 with the phagosome and the role of gp91phox in these interactions (Van Bruggen et al. 2004). A ﬂuorescent fusion protein form of p40phox was used to monitor the association of p40phox with phagosomes in cultured cells (Suh et al. 2006). The ability to express ﬂuorescent forms of components of the NADPH oxidase opens up the possibility of applying advanced optical techniques for the study of the NADPH oxidase in living cells. Other considerations There is some controversy as to whether intracellular site of NADPH oxidase activity, not associated with phagosomes or the plasma membrane, normally exists. This is an important point in understanding the regulation of the oxi- dase and generation of ROS. Therefore, it is important to pay close attention to details of the methodology employed in the various studies that have addressed this point. It should be noted that biochemical assays of production of ROS or the consumption of oxygen in activated neutrophils are typically done with cells in suspension (e.g., Robinson et al. 1984; Dahlgren et al. 2007). Additionally, cell fractionation stud- ies used to track the movement of oxidase components using biochemical assays have neutrophils in suspension as the starting material (reviewed in Nauseef 2004). To further complicate matters, it has been reported that standard cen- trifugation methods used to isolate neutrophils from blood led to the surface expression of molecules that were not detected at the surface of neutrophils in whole blood (Kuijpers et al. 1991). However, most immunocytochemical studies as well as live cell microscopy studies where ROS production is localized occur in cells attached to a substra- tum (e.g., cover slips, microscope slides, or plastic dishes that may also be coated with a variety of proteins) (e.g., DeLeo et al. 1999; Kindzelskii and Petty 2002). As we have already noted, it has been shown that attachment of neu- trophils (and other cells) to surfaces alters their shape; in addition, attachment can modulate their activity with regard to ROS production (e.g., Nathan 1987: Suchard and Boxer