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

Area of Science:

• Immunology
• Cell Biology
• Biophysics

Background:

• Mast cells play a crucial role in allergic responses and inflammation.
• Visualizing serotonin (5-hydroxytryptamine, 5-HT) secretion from mast cells has been technically challenging.
• Understanding mast cell secretion dynamics is key to developing targeted therapies.

Purpose of the Study:

• To image and quantify the secretion process of serotonin (5-HT) from the RBL-2H3 mucosal mast cell line.
• To overcome the technical limitations of deep UV optics in imaging 5-HT autofluorescence.
• To analyze the kinetics and spatial characteristics of 5-HT release during cellular stimulation.

Main Methods:

• Utilized infrared three-photon excitation (3PE) microscopy to image serotonin autofluorescence.
• Analyzed prestimulation 5-HT distributions in loaded and unloaded RBL-2H3 cells.
• Quantified 5-HT release by comparing imaging data with high-performance liquid chromatography (HPLC) measurements.
• Observed antigenically stimulated cells for characteristic ruffling, spreading, and 5-HT disappearance.

Main Results:

• Mast cell stimulation led to granular 5-HT disappearance without detectable granule movement.
• Secretion kinetics were highly heterogeneous, often occurring in quasi-periodic bursts.
• Neighboring granule disappearances were correlated, suggesting localized signaling or granule interactions.
• Residual fluorescence indicated 5-HT remaining bound to the granule matrix in some events.
• Terminal secretion stages involved unresolved granules and leakage of remaining 5-HT.

Conclusions:

• Three-photon excitation microscopy enables direct visualization and quantification of mast cell serotonin secretion.
• Mast cell secretion is a dynamic, heterogeneous process with distinct kinetic patterns.
• The findings provide new insights into the mechanisms of mast cell degranulation and mediator release.