Data processing

Fluorescent images acquired either with the CCD camera or with the confocal microscope were processed with a MetaMorph Imaging System (Universal Imaging Corporation, West Chester, PA, USA). To exclude variations in fluorescent intensity, the signals were corrected for changes in dye concentration by displaying the images in terms of:

F_rel = [(f_t-f_t0)/f_t0]

(in Figures referred as Relative fluorescence), where f_t is the recorded fluorescence and f₀ is the mean of 5-8 sequential frames before UV irradiation with no apparent Ca2+ activity. Moreover, in order to get insight into the dynamics of the intracellular Ca2+ increase, we subtracted preceding images from each other by applying the following equation:

F_inst = [(f_t-f_t-1)/f_t-1]

(in Figures referred as Instant fluorescence), as used by Kidd and Thorn (2000). This procedure allowed the visualization frame by frame of the instantaneous rise in [Ca2+]i and, therefore, to follow the propagation of the signal only in the areas where the Ca2+ release was occurring. To visualize the differences in the fluorescence the custom-designed 6 or 8 grade look up table (LUT) was applied to the 8 bit fluorescent images. To obtain a numerical equivalents of the fluorescence intensity, the average of the intensity measures within the regions of interest (ROIs) was transferred in electronic tables of the Microsoft Excel software and the Relative or Instant fluorescence were calculated as descrived above. The ROIs positioned as indicated in the Figures. The figures for publications were prepared using MetaMorph and Adobe Photoshop software.