Quantifying Calcium Imaging in Neurons

Raw TIFFs were exported and analyzed with ImageJ as previously described^{28 (link)}. An experimenter manually traced the activated cells and determined cell size and relative fluorescent intensity off-line after completing the study. Briefly, Small, medium, and large diameter neurons were defined as having somal areas of <450 μm², 450–700 μm², and >700 μm², respectively. The average fluorescence intensity in the baseline period was taken as F0 and measured as the average pixel intensity during the first two frames of each imaging experiment. The maximum fluorescence intensity, Ft, was measured by calculating the average (peak − background) pixel values in a given region of interest for each image frame recorded during a time interval before and during the stimulation period. The Ft was then used to calculate ΔF/F using the formula ΔF/F = (Ft − F0)/F0. We used ImageJ or Fiji (National Institutes of Health) and LIF (Leica Microsystems GmbH) to analyze calcium imaging data using standard functions and a custom macro. An activated neuron to the stimulation was defined by Ft/F0 > 1.2, as that shown in previous studies^{26 (link)–28 (link)}.