Observation of calcium microdomains at the uropod of living morphologically polarized human neutrophils using flash lamp-based fluorescence microscopy.

The present study outlines improved strategies for ratiometric imaging of cell calcium using a flash lamp-based excitation method and its application to neutrophil polarization. A brief (approximately 6 micros) and intense flash was used to excite the Fluo-4 and Fura Red calcium dye combination in morphologically polarized human neutrophils. These illumination conditions do not allow the dye or calcium ions to diffuse significant distances during the exposure period. Buffer conditions such as pH, pyruvate concentration, and glucose levels were adjusted to more faithfully replicate these parameters in sepsis patients. Fluorescence images at both dyes' emission wavelengths were simultaneously collected using a Dual-View apparatus and an ICCD camera. The ratiometric images, when viewed as single frames or averaged image stacks, clearly demonstrated high calcium probe ratios at the uropod and comparatively low ratios at the cell body that were not evident using conventional imaging methods with longer exposure times. Calcium signaling at the uropod is likely associated with cytoskeletal remodeling during cell motility. (c) 2008 International Society for Advancement of Cytometry