MEMBRANE-PROXIMAL CALCIUM TRANSIENTS IN STIMULATED NEUTROPHILS DETECTED BY TOTAL INTERNAL-REFLECTION FLUORESCENCE

A novel fluorescence microscope/laser optical system was developed to measure fast transients of membrane-proximal versus bulk cytoplasmic i ntracellular calcium levels in cells labeled with a fluorescent calciu m indicator. The method is based on the rapid chopping of illumination of the cells between optical configurations for epifluorescence, whic h excites predominantly the bulk intracellular region, and total inter nal reflection fluorescence, which excites only the region within simi lar to 100 nm of the cell-substrate contact. This method was applied t o Fluo-3-loaded neutrophils that were activated by the chemoattractant N-formyl-met-leu-phe. Chemoattractant-activated cells showed 1) trans ient increases in both membrane-proximal and bulk cytosolic Ca2+ that peaked simultaneously; 2) a larger fractional change (20-60%) in membr ane-proximal Ca2+ relative to bulk cytosolic Ca2+ that peaked at a tim e when the main Ca2+ transient was decreasing in both regions and that persisted well after the main transient was over. This method should be applicable to a wide variety of cell types and fluorescent ion indi cators in which membrane-proximal ionic transients may be different fr om those deeper within the cytosol.