Chemiluminescence microscopy reveals functional heterogeneity in single neutrophils undergoing oxygen burst

Activated polymorphonuclear leukocytes (PMN) respond to various triggers with an oxygen burst, during which the release of reactive oxygen species (ROS) plays a key role in microbial killing. The biological function of the ROS-associated light emissions is not known. However, this particularly weak cell-derived chemiluminescence (CL) may serve as a parameter for the identification of PMN activation. In this study we describe a novel technique which we termed CL microscopy. A microscope-based low-light image-processing system was applied which was sensitive enough to detect single photons, capable of two-dimensional signal accumulation, and digital image analysis. This technique permitted, for the first time, the visualization of the oxygen burst in single cells. Furthermore, quantitative evaluation of cell-derived luminol-enhanced CL revealed functional heterogeneity. Single-cell investigations of activated living PMN of normal human donors showed clear differences in kinetics and intensity of the oxygen burst related to different stimuli. The chemical agent (phorbol 12-myristate 13-acetate) induced CL in 83% of PMN. In contrast, the complement-mediated phagocytic stimulation by opsonized zymosan gave much higher light intensities of individual cells, but only in part of the PMN population (30%). CL microscopy presents a new and highly sensitive technique with considerable potential for single-cell analysis in immunological research.