Table 3.

ImageJ Macros used:

Macrol: Pre-filtering for dot co-localization
showMessage(“Open Channel Red”); //Select Channel Red
open();
red = getTitle();
showMessage(“Open Channel GFP“); //Select Channel GFP
open();
gfp = getTitle(); //Then, we are going to filter both channels
selectWindow(gfp);
run(“Median…”, “radius=1 stack”); //Filtering
run(“Unsharp Mask…”, “radius=2 mask=0.5 stack”); //Filtering
selectWindow(red);
run(“Median…”, “radius=1 stack”); //Filtering
run(“Unsharp Mask…”, “radius=2 mask=0.5 stack”); //Filtering
run(“JACoP ”); //Open JACoP plugin
Macro2: Analyze dots
showMessage(“Open the Image”); //Select image
open();
run(“Duplicate…”, “ ”); //We want to do the segmentation in a copy of the original, therefore we duplicate
run(“Median…”, “radius=1”); //Filtering
run(“Unsharp Mask…”, “radius=2 mask=0.50”); //Filtering
run(“Threshold…”); // to open the threshold window if not opened yet
waitForUser(“Set the threshold and press OK, or cancel to exit macro”); // pauses the execution and lets you access ImageJ manually
run(“Analyze Particles…”); //to take the ROIs
waitForUser(“Finally, use these ROIs in the original window”); //A note to correctly continue after the macro
Macro3: Counting colonies
showMessage(“Open the Image”); //Select image with colonies
open();
rename(“Initial”);
run(“Duplicate…”, “ ”);
run(“Threshold…”); // to open the threshold window if not opened yet
waitForUser(“set the threshold and press OK, or cancel to exit macro”); // pauses the execution and lets you access ImageJ manually as long as you donť press OK, which resumes the macro execution
run(“Convert to Mask”); // to binarize, if you use this command, donť press ‘Apply’ in the threshold window
run(“Watershed”); //to separate close colonies
rename(“Mask”);
waitForUser(“Calibration: Draw a line along the Petri dish \nand introduce the known size in Analize/SetScale, then press OK”);
run(“Threshold…”);
waitForUser(“Set the threshold and press OK, or cancel to exit macro”); // pauses the execution and lets you access ImageJ manually
run(“Analyze Particles…”); //to take the ROIs