Multiphoton imaging of serotonin release

In the figure below an example RBL-2H3 cell is undergoing degranulation in response to an antigenic stimulus. (For the stimulation, multiply conjugated DNP-BSA is used to cross-link cell surface IgE receptors sensitized with anti-DNP IgE.) In the time series of images shown the granular fluorescence (determined as that which is greater than 5 times the basal fluorescence mean) is enclosed by constructed red isocontours. Corresponding plots of the total granular fluorescence (black) and the disappearance rate of that area (red) are shown below. Spatially and temporally correlated release is evident. Multiple granules disappear first at the lower left and a few frames later at the upper right, consistent with a compound exocytosis or cumulative fusion mechanism of secretion. The release rate appears to be oscillatory with a time scale of 1-2 min., the same time scale as measured calcium oscillations that occur in this cell type uppon stimulation. Because MPE microscopy possesses intrinsic 3-d sectioning properties and unparalleled optical penetrability into thick tissue, this technique possesses clear potential for extension into more complex biological systems.