// Displays the colocalization of two images and then measures the morphometrics of the
//overlapped signal
// Installation notes:
// Download and Install WCIF ImageJ package from
// http://www.uhnresearch.ca/facilities/wcif/fdownload.html
// Save this code as .txt file and move file to >Program Files>ImageJ>macros
// Make sure it's the correct ImageJ Folder (the WCIF version) if you have multiple versions
// open WCIF ImageJ and install the macro by selecting from the ImageJ toolbar:
// Plugins>Macros>Install… and then select the .txt file saved earlier.
// Push F8, a window will appear to select a file.
// First select the red image
// another window will appear, now select the green image
// ImageJ will then merge the two images and present a threshold image of the colocalized
// points. There will also be the original images as well as merge between the two with
// colocalized points as white. The image to be analyzed will be autoselected.
// Draw around the area of the image to be analyzed. Only points within this area
// will be measured
// Push F10. ImageJ will perform the measurements and report the results in a new window.
// After copying the results, push F11 to close all windows.
// Push F9 and repeat
// Code below
macro “Close All Windows [F11] ” //F11 used as a shortcut to close all active windows
 {
 while (nImages>0) {
 selectImage(nImages);
 close();
 }
 }
macro “Open Images for colocalization [F8] ” //F8 used as shortcut
{
open(); //select red image
run(“8-bit”); //makes image 8-bit
open(); //select green image
run(“8-bit”); //makes images 8-bit
run(“Colocalization Highligter”, “ratio=50 threshold_channel_1=52 threshold_channel_2=52 display=255
colocalized”); //Colocalization Highlighter is a tool from the WCIF version of
//ImageJ, adjust threshold here if necessary
//user may wish to use this image for making figures
setTool(3); //activates freehand selection tool for drawing around region of interest
selectWindow(“Colocalized points (8-bit) ”); //selects image containing only colocalized
//signals
setThreshold(15, 255); //threshold setting not important here
}
macro “Measure Morphometrics [F10] ” //F10 used as shortcut
{
run(“Clear Results”);
 run(“Set Measurements… ”, “area perimeter circularity redirect=None decimal=2”);
 run(“Analyze Particles… ”, “minimum=5 maximum=500 bins=100 show=Outlines display summarize”); //set size
range of particles here
 for (i=0; i<nResults; i++){
 MP += getResult(‘Perim.’,i);
 MA +=getResult(‘Area’, i);
 MC +=abs(getResult(‘Circ.’, i)); //old version of ImageJ reports circularities of -1 if the
//calculated circularity > 1, so absolute value needed here
 }//loop to assign variables from results
 {
 AMP= (MP/i); //calculates mean perimeter
AMA= (MA/i); //calculates mean area
 AMC= (MC/i); //calculates mean circularity
 Rmorph= (AMA/AMP); //measures the area perimeter ratio, a.k.a. interconnectivity
 print(getTitle());
 print(“Count:” +i);
 print(“Total Area:” +MA);
 print(“Avg. Perimeter:” +AMP);
 print (“Avg. Area:” + AMA);
 print (“Avg. Circularity:”+ AMC);
 print (“Area/Perim:”+ Rmorph);
 selectWindow(“Results”);
 selectWindow(“Log”);
 }
}