ImageJ quantification of fluorescence.

The z-stacks were analyzed four ways using raw data files in ImageJ. Initially, quantification of mean intensity (CrhR/Chl a ratio) and average particle size (μm²) was performed using the histogram and analyze particle functionalities, respectively, in the Fiji package (version 1.53C). Graphs depict analysis of medial slices from 10 independent cell fields, comprising a minimum of 300 cells. Then, to depict the relative transverse localization of red autofluorescence and green CrhR-derived fluorescence, transects were drawn to encompass Chl a (red) and the predominant CrhR (green) signals in representative cells and analyzed using ImageJ Plot Profile. All CrhR pixel values were normalized (y = 1.0) to the point of maximum measured arbitrary units. Finally, surface plot profiles were generated using the Interactive 3D Surface Plot plugin (version 2.4.1) on ovals encompassing representative cells.