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This experiment involves measurement of neurotransmitter release from cultured hippocampal neurons. The neurotransmitter-containing vesicles in the neurons have been stained with a fluorescent dye called FM1-43. Electrical stimulation of the neurons causes these vesicles to fuse with the plasma membrane and the fluorescence signal decreases as the dye is released. We are interested in the kinetics of the dye release.

The panel has various options which allow you to point at any number of experiments, each of which contains {a set of fluorescence images taken at different times, a phase-contrast image of the same field of neurons, and a file indicating the timing of the fluorescence images}. The background is subtracted, the images are aligned to each other, the sites of transmitter release are found by an algorithm that searches for local fluorescence maxima according to size and intensity spatial derivative, the destaining of each of the sites is determined against time, this destaining is scored according to several user-weighted factors for "quality", and the destainings exceeding a certain quality threshold are averaged. All of this is done without user intervention except for the ability to manually remove spuriously found release sites.

The graphs called "QualityMapMain" and Graph1 are produced automatically. In the quality map, release sites are color coded acording to the quality score described above and overlayed on the phase contrast image of the neurons. Blue sites are "good" and red are "bad", the brighter they are the farther from they are from the threshold. In Graph1 the upper trace shows the timing and frequency of stimulation. For demonstration purposes I have made some other graphs: Graph6 contains the user-accepted (white) and user-rejected (red) automatically found release sites overlayed on a green image of FM1-43 fluorescence. Graph3 shows the same but the overlay is on a phase contrast picture. All of these graphs are abritrarily zoomed in to show detail.

Submitted by Jeremy Bergsman of Yale University.