// Processing raw data with image correction images from the microscope for TRUE background-subtracted biological signal.
// Followed by mask generation and application to the Signal channel to measure mean of single cells.
// This macro was written by Darren Thomson February 2018. Any comments or quereys should be directed to Darrenthomson.research@gmail.com

// Instructions: Make folders for GFP and RFP images (copy them into the folder). Also make an 'output' folder to store processed images and results. 
// Open the 'Dark' and MedianShade' images in FIJI.
// run this macro and locate the folders for  GFP, RFP and output. 
// Leave the macro to run, DO NOT OPEN OR SELECT ANYTHING ELSE (IN FIJI) WHILE THE MACRO IS RUNNING
// After it has finished, save the final results from all the images as .xls
// IMPORTANT. Quality control Look at the output folder and check that the macro has perfomed to a satisfactory level.
// Here you can identify big mistakes, relate it to a unique cell # and take them out of the data.


//What does the macro do? 
// Sequentially opens a set of GFP/RFP images 
// The GFP and RFP images will be background-corrected followed by mask generation in the GFP channel that is then applied to the RFP signal image for measuring. 
//A results table will appear at the end which is saved containing measurements from each individual object (cell) 

// Problems to solve: 1) Analyze particles does not segment crossing hyhae and just counts one large item.

//Step 1 Image correction for background camera and microscope noise/illumination

// The small macro below utilises data with 2 channels, put into different folders. It will find the file in one folder and look for a similar named file, but with the channel descriptor, in another folder. 


dir1 = getDirectory("Where are the GFP images"); // This is where the Ch1 images are
list = getFileList(dir1); // This lists all the files in Ch1 folder
dir2 = getDirectory("Where are the RFP images"); // This is where the Ch2 images are
SaveDir = getDirectory("Where to save processed images + results"); // This is where results/processed images are stored
for (g=0; g<list.length; g++) { // This goes through each file in Ch1 iteratively
ch1name = list[g]; // This takes the name of the file and makes it a variable called ch1name
ch2name = replace(ch1name, "w2GFP", "w3RFP"); // This generates Ch2name variable by replacing IDs 
if (File.exists(dir2 + ch2name)); { // This looks in the Ch2 folder for files matching ch2name. If there is, then it enters the loop. 

  print(ch1name);
  open(dir1 + ch1name);
  rename ("GFP"); // This renames the images to something simple for the image calculator below
 print(ch2name);
  open(dir2 + ch2name);
  rename ("RFP"); // This renames the images to something simple for the image calculator below

// Above I renamed the images 'GFP' and 'RFP' for the image calulator to work below.

imageCalculator("Subtract create 32-bit", "GFP","Dark.tif");
rename ("GFPSub");
imageCalculator("Divide create", "GFPSub","MedianShade.tif");
rename ("GFPcorrected");
selectWindow("GFP");
close();
selectWindow("GFPSub");
close();
selectWindow("GFPcorrected");
run("16-bit");
selectWindow("GFPcorrected");
title = getTitle;
saveAs("tiff", SaveDir+ch1name+title); // This needs to know the images' original name to save.

imageCalculator("Subtract create 32-bit", "RFP","Dark.tif");
rename ("RFPSub");
imageCalculator("Divide create", "RFPSub","MedianShade.tif");
rename ("RFPcorrected");
selectWindow("RFP");
close();
selectWindow("RFPSub");
close();
selectWindow("RFPcorrected");
//run("16-bit"); // THIS IS A BUG IT MAKES THE -ve control VALUES MUCH HIGHER AFTER 16 BIT CONVERSION
selectWindow("RFPcorrected");
title = getTitle;
saveAs("tiff", SaveDir+ch2name+title); // This needs to know the images' original name to save.

// close(ch1name)
 //close(ch2name) 
  
// Step 2: generate ROIs from the GFP channel

ids=newArray(nImages); //checks how many images are currently open
n=nImages;
for (i=0;i<nImages;i++) { 
        selectImage(i+1); 
        title = getTitle; // registers the names of all open images
if (endsWith(title, "GFPcorrected.tif")){
         run("Subtract Background...", "rolling=45");
	run("Gaussian Blur...", "sigma=3");
	setAutoThreshold("Huang dark");
	//run("Threshold...");
	setOption("BlackBackground", false);
	run("Convert to Mask");
	//run("Watershed");
	run("Set Measurements...", "area mean standard modal min centroid feret's integrated median stack display redirect=None decimal=3");
	run("Analyze Particles...", "size=1500-Infinity circularity=0.0-0.7 add"); 
	//run("Create Selection"); // This Creates a mask outwith analyze particles, which will not have inclusive holes, but only the hyphae (but also unfiltered particles).
	//run("Make Inverse"); // these 3 commands are useful if you ever wanted to compare the signal inside vs outside the mask. This creates the inverse of the masks and adds it to the list of ROIs. 
	//roiManager("Add"); // This adds the 'total' mask to be measured.	
if (endsWith(title, "GFPcorrected.tif")){
         saveAs("tiff", SaveDir+ch1name+"mask");
}
		}
}	

for(h=0; h<roiManager("Count"); h++){
roiManager("Select", h);
//Rename nuclei
roiManager("Rename", "Sulphydrated_FungalCell"+(h+1));
}

// Step3: apply the ROIs to the RFP channel

ids=newArray(nImages); //checks how many images are currently open
n=nImages;
for (i=0;i<nImages;i++) { 
        selectImage(i+1); 
        title = getTitle;

		if (endsWith(title, "RFPcorrected.tif")){ //HERE YOU NEED to adjust the descriptor of your target channel. It selects it so that the ROI manager puts its ROIs onto the target channel.
         roiManager("Show All"); // this applies the masks to the target channel
		roiManager("Measure");
		roiManager("reset"); // If you don't reset the ROI manager will accumulate ROIs from each dataset that's loaded in.
		selectWindow("Results"); // This just puts the results window at the foreground of the desktop
		saveAs("Measurements", SaveDir+ch1name +"-RFPResults.csv"); // This saves the results as a meaningful name, else it will be re-written each time.
		close();
		}

}	
	}
}
ids=newArray(nImages); //checks how many images are currently open
n=nImages;
for (i=0;i<nImages;i++) { 
        selectImage(i+1); 
        title = getTitle;
        
        selectWindow("Results"); // This just puts the results window at the foreground of the desktop
		saveAs("Measurements", SaveDir+"ALL-RFPResults.csv"); // This saves the results as a meaningful name, else it will be re-written each time.
}