CONFOCAL RAMAN MICROSPECTROSCOPY OF THE ACTIVATION OF SINGLE NEUTROPHILIC GRANULOCYTES

Confocal Raman micro-spectroscopy has been applied to investigate the activation process of single, living neutrophilic granulocytes. Both r esting cells as well as activated cells were measured. The activation of cells was performed with phorbol-l 2-myristate-13-acetate activator and Escherichia Coli bacteria. Raman microspectroscopy combines a hig h spatial resolution inside a single, living cell with detailed materi al information. Using this approach it can be concluded that activatio n of the cells with phorbol-12-myristate-13-acetate causes a change in the redox state of cytochrome b(558). This protein is a part of the N ADPH-oxidase complex that neutrophilic granulocytes employ to generate O-2(-), superoxide anion. Additionally a change in the redox state of myeloperoxidase can be observed. Myeloperoxidase is known to react wi th O-2(-). Activation of the cells with bacteria gives rise to corresp onding changes in the Raman spectra. From this single cell study it ca n be concluded that the enzymes cytochrome b(558) and myeloperoxidase are present inside the cytoplasm of the living fell, while participati ng in the redox processes. Activation causes an intra-cellular release of oxygen metabolites. Activation with bacteria of neutrophilic granu locytes from a patient with chronic granulomatous disease, that contai n no cytochrome b(558), led to typical changes in the redox state of m yeloperoxidase. This indicates that in the bacterium/neutrophilic gran ulocyte system oxygen metabolites are generated that are capable of re acting with MPO.