Gm. Omann et D. Axelrod, MEMBRANE-PROXIMAL CALCIUM TRANSIENTS IN STIMULATED NEUTROPHILS DETECTED BY TOTAL INTERNAL-REFLECTION FLUORESCENCE, Biophysical journal, 71(5), 1996, pp. 2885-2891
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.