DEVELOPMENT OF VIDEO MICROIMAGE ANALYSIS AND ITS APPLICATION FOR THE DYNAMICS OF A SINGLE NEUTROPHIL RESPONSE

The real time sequence of phagocytosis-exocytosis in a single neutroph il was directly visualized by video-enhanced contrast differential int erference contrast (VEC-DIC) microscopy. Exocytotic responses were inv estigated with special reference to their dynamic and spatio-temporal properties. By using a dual imaging microscope that allowed one to obs erve DIC and fluorescence images simultaneously at high magnification, exocytotic responses and cellular calcium concentration ([Ca2+](i)) w ere directly investigated. [Ca2+](i) transients were more closely rela ted to the extension of pseudopodia for engulfing a foreign particle a nd not directly to exocytosis. A chemiluminescence microscope equipped with a photon-counting camera was further applied for the detection o f active oxidants. The production of active oxidants during phagocytos is was detected at the sub-cellular level. The chemiluminescence was o bserved exclusively in the region of the phagosome, suggesting that ac tive oxidants involved in killing of foreign particles were released d irectly into the phagosome. Superoxide anions (O-2(-)) generated by st imulated neutrophils were specifically detected and quantified. One co unt obtained by this system was equivalent to 59 amol of O-2(-). Maxim um O-2(-) production was observed at 6 similar to 8 min after stimulat ion and was estimated at 1.9 fmol/min/cell on average.