Comments

// Comments allow you to put human-readable thoughts
// into your code.

// The goal of this "macro" is simply to teach you about comments!

// Comments help you to remember why you did something:
// Set the value to "2" because my boss said so!
value = 2; // Comments can be added to any line!

// Code can be disabled by commenting it out:
// x = y * 2;
					

Variables

What is a variable?

• Open "Blobs" image (File > Open Samples > Blobs (25K))

run("Blue");
run("Fire");
					

Variables

What is a variable?

• Container
• "A piece of code that can vary"

color = "Blue";
run(color);
					

• Two Types: Numbers or Strings

Variables

// what sorts of values can we assign?
title = "Hello, World!";                // string
intensity = 255;                        // number
a = exp(x * sin(y)) + atan(x * y – a);  // expression

// string constant vs. variable name
text = "title"; 
text = title;

x = 3;
y = x;
x = 5; // what is the value of y after this?

// the variable is assigned after the expression is evaluated
intensity = intensity * 2;
					

Strings

name = "copy";

// you can concatenate strings, and strings and numbers
text = "The name is " + name;

// numbers versus strings and more concatenation...
a = 2;
b = 3;
print(a + b);

a = "2";
b = "3";
print(a + b);

// what happens when we run this?
run("Duplicate...", "title=name");

// what's different with this line?
run("Duplicate...", "title=" + name);
					

Functions

function myFunction(arguments) {
	// Your code goes here
}

					

function setLUT(lutName) {
	run(lutName);
}

					

Functions

print("Hello, world!");

// functions can return values
// Hint: use parameters instead of calling getNumber
number = getNumber("Type in a number!", 5);

// the "run" function is the most important one
// it follows the structure of run("Command...", "Argument(s) String");
run("Duplicate...", "title=New");

// for arguments with spaces, enclose in square brackets
run("Duplicate...", "title=[with spaces]");
					

Conditionals

#@String instructor

if (getBoolean("Is " + instructor + " going too fast?")) {
    hint = "Tell them to to slow down!";
}
else {
    hint = "Try to modify the code, play with it...";
}
showMessage("Advice:", hint);
					

Loops

// For loops:
// They use assignment, test, increment
// Use when you know the # of times the loop will run
for (i = 1; i <= 10; i++) {
    print("Counter: " + i);
}

// While loops:
// Use when you do not necessarily know the # of times the loop will run
while (getBoolean("Do you want me to keep going?")) {
    print("Ok, I'm still going...");
}
showMessage("Ok, I'm done!");
					

Tying it all together

Do you notice a bug in this code?

// this example makes a stack of blurred versions of the
// current slice with a range of radii.

radius = getNumber("Maximal radius?", 5);

title = "Blurred stack of " + getTitle();
run("Duplicate...", "title=[" + title + "]");
run("Select All");
run("Copy");
for (i = 1; i <= radius; i++) {
        run("Add Slice");
        run("Paste");
        run("Gaussian Blur...", "radius=" + radius);
}
					

1. Launch the Recorder

• Use the Search Bar! (Ctrl+L)
• OR: click the Dev icon, then Record...
• OR: Plugins ▶ Macros ▶ Record...

2. Execute operations

• Open sample images
• Download/unpack "images/C3-jw-30min.zip"
• Plugins ▶ Bio-Formats ▶ Bio-Formats Importer
• Open "C3-jw-30min 5_c5.tif"
• Apply Gaussian blur
• Apply a threshold (Shift+T)
• Create Mask
• Watershed
• Analyze Particles...
  • Size: 200-Infinity
  • Exclude on edges; Add to Manager

Code Check!

run("Gaussian Blur...", "sigma=2");
setAutoThreshold("Default dark");
//run("Threshold...");
run("Create Mask");
run("Watershed");
run("Analyze Particles...", "size=200-Infinity exclude add");
					

3. Spruce it up

• Click the "Create" button
• Use script parameters
• setBatchMode(true)
• Use image IDs
• Store in scripts
  • In a folder matching the desired menu
  • Use underscore for spaces

Code Check!

#@int(label = "Minimum size") minSize

setBatchMode(true);
id = getImageID(); // get original image id
run("Duplicate...", " "); // duplicate original image and work on the copy
run("Gaussian Blur...", "sigma=2");
setAutoThreshold("Default dark");
//run("Threshold...");
run("Create Mask");
run("Watershed");
run("Analyze Particles...", "size=" + minSize + "-Infinity display exclude add");
run("Clear Results"); // delete contents of results table
selectImage(id); // activate original image
roiManager("Show All with labels"); // overlay ROIs
roiManager("Deselect");
roiManager("Measure"); // measure on original image
					

Example script to count nuclei in multiple images in a folder/subfolders:

/*
* Macro to count nuclei in multiple images in a folder/subfolders.
*/

#@File(label = "Input directory", style = "directory") input
#@File(label = "Output directory", style = "directory") output
#@String(label = "File suffix", value = ".tif") suffix
#@int(label = "Minimum size") minSize

processFolder(input);
	
// function to scan folders/subfolders/files to find files with correct suffix
function processFolder(input) {
	list = getFileList(input);
	for (i = 0; i < list.length; i++) {
		if(File.isDirectory(input + File.separator + list[i]))
			processFolder("" + input + File.separator + list[i]);
		if(endsWith(list[i], suffix))
			processFile(input, output, list[i]);
	}
	//saves results for all images in a single file
	saveAs("Results", output + "/All_Results.csv"); 
}

function processFile(input, output, file) {
	setBatchMode(true); // prevents image windows from opening while the script is running
	// open image using Bio-Formats
	run("Bio-Formats", "open=[" + input + "/" + file +"] autoscale color_mode=Default rois_import=[ROI manager] view=Hyperstack stack_order=XYCZT");
  id = getImageID(); // get original image id
	run("Duplicate...", " "); // duplicate original image and work on the copy
	
	// create binary image
	run("Gaussian Blur...", "sigma=2");
	setAutoThreshold("Default dark");
	//run("Threshold...");
	run("Create Mask");
	run("Watershed");
	// save current binary image
	save(output + "/Binary_OUTPUT_" + file);
	
	run("Analyze Particles...", "size=" + minSize + "-Infinity exclude add");
  selectImage(id); // activate original image
  roiManager("Show All with labels"); // overlay ROIs
	roiManager("Deselect");
	roiManager("Measure"); // measure on original image
	
	// save ROIs for current image
	roiManager("Deselect");
	roiManager("Save", output+ "/" + file + "_ROI.zip"); // saves Rois zip file
	roiManager("Deselect");
	roiManager("Delete"); // clear ROI Manager for next image
}