Histochem Cell Biol (2008) 130:281–297 291 Fig. 7 Electron micrographs of thick sections (0.35 lm thickness) of human neutrophils incubated for the cytochemical detection of alkaline phosphatase. a An unstimulated cell that was incubated for the detection of alkaline phosphatase activity in the absence of cell permeabilization with Triton X-100. Note that there is no reaction in the absence of permeabilization. b A cell that was incubated for the detection of alkaline phosphatase activity following cell permeabili- zation. In this case, numerous small rod-shaped organelles containing reaction product are evident. c A cell stimulated with f-Met-Leu-Phe (FMLP) and cytochalasin B (Cyt.B) that was incubated for the detection of alkaline phosphatase activity in the absence of cell- Biophysical methods for study of the phagocyte NADPH oxidase A chemiluminesence signal was detected during phago- cytosis and was thought to be associated 1O2 generation (Allen et al. 1972). Luminol was incorporated into these studies later to enhance the chemiluminesence signal (Allen and Loose 1976). Subsequently, real-time visuali- zation of chemiluminescence associated with the respiratory burst was reported in a series of studies (Suematsu et al. 1987, 1989; Suematsu and Tsuchiya 1991). For example, rabbit neutrophils were used to study permeabilization. Note the dramatic rearrangement of the alkaline phosphatase compartments into elaborate tubular structures. These tubular structures appear to be in continuity with the extracellular space since permeabilization was not required to detect the enzyme activity. d A cell stimulated with f-Met-Leu-Phe (FMLP) and cytochalasin B (Cyt.B) that was incubated for the detection of alkaline phosphatase activity in the presence of cell permeabilization. This cell has a appearance similar to the cell treated in the same manner but without permeabilization. The cytochemical reaction was carried out as we have described previously (Kobayashi and Robinson 1991). Bars: 1 lm exocytosis associated with formation of the phagosome in the presence of luminol (Suzaki et al. 1997). These studies required the use of a very sensitive photon-counting cam- era. Most of the recorded photons were in the vicinity of particles in the phagosomes. It has been proposed that chemiluminesence has advantages for imaging ROS gene- ration in living cells when compared to ﬂuorescence microscopy (Suematsu and Tsuchiya 1991). The suggested advantages are: (1) since the light is derived from the chemical reaction itself (i.e., chemiluminescence), excita- tion illumination is not required (excitation illumination may alter structure and function in living cells); (2) 123