Volocity software (version 6.6.2; PerkinElmer) was used for image acquisition, and Ca2+ image postprocessing was done with Matlab (MathWorks) (37). First, the background in each channel was corrected. Bleaching of Fura Red was corrected using a frame-by-frame elevation of pixel intensities by the difference between the initial value and the fit value of the specific frame with an additive approach. To obtain digital confocal images, Fluo4 and Fura Red were deconvolved using the Lucy-Richardson algorithm with an analytically computed point-spread function, and both fluorescence channels were automatically aligned by a rigid sum-of-squared-differences–based registration. In a final step, the Fluo4/Fura Red ratio was generated, median filtered (3 × 3), and exported.

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