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. 1999 Apr;76(4):1835–1846. doi: 10.1016/S0006-3495(99)77343-1

Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence.

R M Williams 1, J B Shear 1, W R Zipfel 1, S Maiti 1, W W Webb 1
PMCID: PMC1300160  PMID: 10096882

Abstract

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.

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Selected References

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