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// FISHji4

// LEPABE 09-02-2015

// Daniel Carvalho & Analia Lourenco

// Macro for ImageJ. Determination of the mean fluorescence intensity.

var min_size=0;

var max_size=0;

var min_circ=0;

var max_circ=0;

var distance_pixel=0;

var known_distance=0;

var aspect_ratio=0;

var unit_length="";

var channel = "green";

macro "FISHji4"{

inputFolder = getDirectory("Choose the input folder!");

help= "<html>Search is case sensitive. Regex patterns can also be used, e.g., "

+"\"<tt>[0-9&&[^1]]\</tt>\" to find a digit that is not 1, or \"<tt>-.*-\</tt>\" to "

+"find any character sequence flanked by hyphens. You may need to "

+"escape metacharacters ('.', '[', ']', '^', '$', etc.) by a backslash. "

+"Note that replaced patterns should always give rise to unique "

+"names as the ROI Manager cannot cope with duplicated entries.";

openDialog();

outputFolder = getDirectory("Choose the output folder!");

setBatchMode(true);

images = getFileList(inputFolder);

imageCount = countImages(images);

notice = "converted: \n";

//out = File.open(outputFolder+"statistics.txt");

for (i=0; i<images.length; i++) {

showProgress(i, imageCount);

inputPath = inputFolder + images[i];

if (isImage(inputPath)) {

processFISH(inputFolder,images[i],outputFolder);

outputPath = outputFolder + images[i];

//save(outputPath);

notice = notice + outputPath + "\n";

}

setBatchMode(false);

print(notice);

exit();

function processFISH(inputFolder,image,out) {

inputPath = inputFolder + image;

open(inputPath);

title=getTitle();

run("RGB Split");

selectImage(title+" ("+channel+")");

// Set scale: distance in pixels, known distance, pixel aspect ratio, unit of length;

run("Set Scale...", "distance=distance_pixel known=known_distance pixel=aspect_ratio unit=unit_length global");

run("Subtract Background...", "rolling=80"); // Application of the "Rolling Ball" algorithm

t=getTitle();

run("Duplicate...", "title=roi");

run("Convolve...", "text1=[0 0 0 -1 -1 -1 0 0 0\n0 -1 -1 -3 -3 -3 -1 -1 0\n0 -1 -3 -3 -1 -3 -3 -1 0\n-1 -3 -3 6 13 6 -3 -3 -1\n-1 -3 -1 13 24 13 -1 -3 -1\n-1 -3 -3 6 13 6 -3 -3 -1\n0 -1 -3 -3 -1 -3 -3 -1 0\n0 -1 -1 -3 -3 -3 -1 -1 0\n0 0 0 -1 -1 -1 0 0 0\n] normalize"); // Convolution Laplacian of Guassian kernel 9x9

run("Make Binary"); // automatic threshold;

run("Set Measurements...", " mean redirect=["+t+"] decimal=0"); // Overlay;

run("Analyze Particles...", "size=min_size-max_size pixel circularity=min_circ-max_circ show=Nothing add"); // size and circularity parameters

roiManager("Show all without labels");

run("Colors...","selection=magenta");

open(inputPath);

original=getTitle();

print(">>>>O: "+title);

selectImage("roi");

roi=getTitle();

print(">>>>R: "+roi);

run("Add Image...","image=[&original]");

run("From ROI Manager");

run("Flatten");

saveAs("Tiff",out+"/["+original+"]_1");

run("Close");

run("Clear Results");

close();

}

function generateStatistics(out){

statistics=newArray("Mean");

kk = newArray(statistics.length*nResults);

for (j=0; j<statistics.length; j++){

for (i=0;i<nResults; i++){

kk[j+i*statistics.length] = getResult(statistics[j],i);

}

// Statistics output

Array.getStatistics(kk, min, max, mean, stdDev);

print(out,"\nImage: " + title);

print(out,"Mean: " + mean);

print(out,"STD: " + stdDev);

}

function isImage(filename) {

extensions = newArray("tif", "tiff", "jpg", "bmp");

result = false;

for (i=0; i<extensions.length; i++) {

if (endsWith(toLowerCase(filename), "." + extensions[i]))

result = true;

}

return result;

}

function openDialog(){

Dialog.create("FISH Image Processing");

Dialog.setInsets(12,0,0);

Dialog.addNumber("Minimum image size:", 0);

Dialog.addNumber("Maximum image size:", 500);

Dialog.addNumber("Minimum image circularity:", 0);

Dialog.addNumber("Maximum image circularity:", 1);

Dialog.setInsets(12,0,0);

Dialog.addNumber("Distance in Pixels:", 0);

Dialog.addNumber("Known Distance:", 0);

Dialog.addNumber("Pixel Aspect Ratio:", 1.0);

Dialog.addString("Unit of Length:", "pixel");

Dialog.setInsets(12,0,0);

items_c = newArray("green", "blue", "red");

Dialog.setInsets(1,1,10);

Dialog.addRadioButtonGroup("Channel:", items_c, 3, 1, "green");

help= "<html>"

+"<h3>Description of Parameters:</h3> "

+"Size: Values range from 0 (minimum image size) to infinity (maximum image size). Values are expressed in size square units or pixels. "

+"Circularity: Values [4*pi*(area/perimeter^2)] range from 0 (infinitely elongated polygon) to 1 (perfect circle). "

+"Channel: Select the correct channel (red, green, and blue) for each input folder, images will be separated in order to analyse the channel where fluorescence is emitted. "

+"Further details about the macro and examples can be found at <a href='http://193.137.11.213/fish/'>FISHji's Web site.</a>"

+" </html>";

Dialog.addHelp(help);

Dialog.show();

min_size = Dialog.getNumber();

max_size = Dialog.getNumber();

min_circ = Dialog.getNumber();

max_circ = Dialog.getNumber();

distance_pixel = Dialog.getNumber();

known_distance = Dialog.getNumber();

aspect_ratio = Dialog.getNumber();

unit_length = Dialog.getString();

channel = Dialog.getRadioButton();

}

function countImages(array) {

result = 0;

for (i=0; i<array.length; i++) {

if (isImage(array[i])) result++;

}

return result;

}