//LigninJ Version 1.0
//Author: Masato Nakamura, Tomoaki Kamehama, Yasushi Sato
//Organization: Graduate School of Science and Engineering, Ehime University, Japan
//Source: Masato Nakamura, Tomoaki Kamehama, Yasushi Sato (2020) Image analysis of stress-induced lignin deposition in Arabidopsis thaliana using the macro program LigninJ for ImageJ software. Plant Biotechnology. (https://doi.org/10.5511/plantbiotechnology.20.0110a)

macro "LigninJ" {
requires("1.52q");

d = getDirectory("Choose a Directory");
Dialog.create("Parameters");
Dialog.addNumber("Known distance:", 100);
Dialog.addString("Unit:", "um");
Dialog.addNumber("Pixels of known distance:", 73.9);
Dialog.addNumber("Lower threshold of a* :", 20);
Dialog.addNumber("Lower size limits of lignified area (Unit^2) :", 20);
Dialog.show();
D=Dialog.getNumber();
Unit=Dialog.getString();
P=Dialog.getNumber();
LT=Dialog.getNumber();
LS=Dialog.getNumber();

files = getFileList(d);
   for (i = 0; i < files.length; i++) {
   f = d + files[i];
   doLignin(f);
   }

function doLignin(f) {
setBatchMode("hide");
open(f);

run("Set Scale...", "distance=P known=D pixel=1 unit=Unit global");
rename("copy");
origBit = bitDepth;
if (bitDepth() != 24) exit;

//Reduce picture to 1/50 for background correction
run("Averaging Reducer", "x=50 y=50");
   max = 0;
   width = getWidth(); height = getHeight();
   for (y=0; y<height; y++) {
      if (y%20==0) showProgress(y, height);
      for (x=0; x<width; x++) {
          value = getPixel(x,y);
          if (value>max) {
              max = value;
              xmax = x;
              ymax = y;
          }
      }
   }
selectWindow("Reduced copy");
run("RGB Stack");
val = newArray(3);
for (s=1;s<=3;s++) {
   setSlice(s);
   val[s-1] = getPixel(xmax,ymax);
   }
run("Close");

selectWindow("copy");
run("RGB Stack");
for (s=1; s<=3; s++) {
   setSlice(s);
   dR = 255/val[s-1];
   run("Multiply...","value=dR slice");
   }
run("Convert Stack to RGB");
rename("correct");
newpath = File.directory + File.separator + File.nameWithoutExtension+"_1.jpg";
saveAs(".jpg", newpath);
run("Lab Stack");
run("Next Slice [>]");
run("Threshold...");
setThreshold(LT, 128.0000);
run("Set Measurements...", "area redirect=None decimal=3");
run("Analyze Particles...", "size=LS-infenity summarize add slice");

selectWindow("correct");
newpath = File.directory + File.separator + File.nameWithoutExtension+"_2.jpg";
saveAs(".jpg", newpath);
run("Set Measurements...", "area mean redirect=None decimal=3");
if (roiManager("count") > 0) {
   roiManager("Multi Measure append");
   roiManager("Delete");
   } else {
   makeRectangle(1,1,1,1);
   roiManager("add");
   run("Set Measurements...", "area redirect=None decimal=3");
   roiManager("Multi Measure append");
   roiManager("Delete");
   }
run("Close All");
selectWindow("Threshold");
run("Close");
}

selectWindow("Summary of correct");
newpath = File.directory + File.separator + "Summary.csv";
saveAs("results", newpath);
run("Close");
selectWindow("Results");
newpath = File.directory + File.separator + "Results.csv";
saveAs("results", newpath);
run("Close");
}