Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence.
The secretion process of the mucosal mast cell line RBL-2H3 was imaged using infrared three photon excitation (3PE) of serotonin (5-hydroxytryptamine, 5-HT) autofluorescence, a measurement previously difficult because of the technical intractability of deep UV optics. Images of prestimulation 5-HT distributions were analyzed in loaded cell populations (those incubated in a 5-HT-rich medium overnight) and in unloaded populations and were found to be strictly quantifiable by comparison with bulk population high-performance liquid chromatography measurements. Antigenically stimulated cells were observed to characteristically ruffle and spread as granular 5-HT disappeared with no detectable granule movement. Individual cells exhibited highly heterogeneous release kinetics, often with quasi-periodic bursts. Neighboring granule disappearances were correlated, indicative of either spatially localized signaling or granule-granule interactions. In one-half of the granule release events, weak residual fluorescence was visible suggestive of leftover 5-HT still bound to the granule matrix. The terminal stages of secretion (>300 s) consisted primarily of unresolved granules and remainder 5-HT leakage from already released granules.
Williams
R M
RM
School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, USA. rw36@cornell.edu
Shear
J B
JB
Zipfel
W R
WR
Maiti
S
S
Webb
W W
WW
D000818:Animals; D001693:Biological Transport, Active; D055592:Biophysical Phenomena; D001703:Biophysics; D015550:Cell Degranulation; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism; D007700:Kinetics; D008407:Mast Cells / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods*; D017785:Photons; D051381:Rats; D012701:Serotonin / Q000378:metabolism*
D012701:Serotonin