The script is compatible with MATLAB R2021a and newer versions.

runPipeline_1.m: Specify the number of channels and the folders containing multichannel images (each channel must be placed in a separate directory). Define segmentation settings by selecting: the channel used for segmentation the channel(s) from which fluorescence values are extracted

segmentation parameters

Example — Case 1 (segmentation of nuclei and chloroplasts separately): Parameters used to achieve optimal segmentation:

Nuclei: backgroundOffset = 1 adaptiveSensitivity = 0.6 adaptiveNeighbourhood = 11 howround = 4 sizeMin = 5, sizeMax = 10000

Chloroplasts: backgroundOffset = 1 adaptiveSensitivity = 0.6 adaptiveNeighbourhood = 3 howround = 20 sizeMin = 5, sizeMax = 2000

Cytoplasm: Defined as the inverse mask of the combined nuclei and chloroplast masks. backgroundOffset = 1 (for both segmentation channels) adaptiveSensitivity = 0.6 (both channels) adaptiveNeighbourhood = 11 (mKO2) and 3 (Venus) howround = 20 (both channels) sizeMin = 5, sizeMax = 10000 (both channels)

Example — Case 2 (segmentation of nuclei): backgroundOffset = 20 adaptiveSensitivity = 0.01 adaptiveNeighbourhood = 11 howround = 20 sizeMin = 25, sizeMax = 200

runSegmentation.m: for segmentation of remaining pixels, uncomment lines 443-446: % if segmentation needs to be inverted (0s become 1s and vice versa) for ii = 1:numel(masks) masks{ii} = ~masks{ii}; end