iba1-extraction.Rmd

---
title: "Microglia Channel Extraction"
description: |
 This ImageJ script extracts the IBA1 channel from multi-channel TIFF images and assigns each image a random alphanumeric code for blinded microglia annotation.
author:
  - first_name: "Ayush"
    last_name: "Noori"
    url: https://www.github.com/ayushnoori
    affiliation: Massachusetts General Hospital
    affiliation_url: https://www.serranopozolab.org
    orcid_id: 0000-0003-1420-1236
output:
  distill::distill_article:
    toc: true
---

```{r setup, include = FALSE}
knitr::opts_chunk$set(eval = FALSE)
```

This script is written in the ImageJ Macro Language (IJM).

``` {.ijm .IJM}
macro "IBA1 Channel Extraction" {

	setBatchMode(true);

	input = getDirectory("Choose input data folder.");
	files = getFileList(input);
	// Array.show(files);

	dir = "C:/Users/ayush/Dropbox (Partners HealthCare)/Single-Cell Multiplex IHC/Multiplex IHC Project/Multiplex IHC (GitLab)/";
	datadir = dir + "Data/2 - Channel Extraction/Microglia/";


	////////////////////////////////////////////////////////////
	/////  FUNCTION FOR RANDOM ID
	////////////////////////////////////////////////////////////
	
	function randomString(length, chars) {
		result = "";
		for (i = 0; i < length; i++) {
			maxlen = lengthOf(chars)-1;
			rand = round(random * maxlen);
			result +=  substring(chars, rand, rand+1);
		}
		return result;
	}
	

	////////////////////////////////////////////////////////////
	/////  ITERATE OVER IMAGES
	////////////////////////////////////////////////////////////
	
	for (f = 0; f < files.length; f++) {

		open(input + files[f]);
		Roi.remove; // remove active selection, if any
	
		////////////////////////////////////////////////////////////
		/////  LOAD IMAGE + DEFINE MARKERS
		////////////////////////////////////////////////////////////
		
		image = getTitle(); // get crop title
		selectImage(image); // shift focus to the selected crop
		filename = substring(image, 0, indexOf(image, "_Reordered.tif"));
		
		splitname = split(filename, "_");	
		sample = splitname[0];
		layer = splitname[1];
		crop = splitname[2];
		crop = substring(crop, 4);
		
		if (sample == "1190" || sample == "1301" || sample == "1619" || sample == "2169" || sample == "2191" || sample == "2250" || sample == "2274") {
			condition = "CTRL";
		} else {
			condition = "AD";
		}
		
		print(filename);

		////////////////////////////////////////////////////////////
		/////  RANDOMIZE CROP
		////////////////////////////////////////////////////////////

		id = randomString(6, "0123456789abcdefghijklmnopqrstuvwxyz");
		// print(id);

		Table.set("ID", f, id);
		Table.set("Sample", f, sample);
		Table.set("Layer", f, layer);
		Table.set("Crop", f, crop);
		Table.set("Condition", f, condition);
		Table.set("File", f, filename);
		Table.update();
	
		////////////////////////////////////////////////////////////
		/////  SAVE IBA1 CHANNEL
		////////////////////////////////////////////////////////////
		
		// duplicate IBA1 channel
		selectImage(image); // shift focus to original
		run("Duplicate...", "title=IBA1 duplicate channels=3");

		run("Enhance Contrast...", "saturated=0.1"); // only for visualization purposes
		
		selectWindow("IBA1"); // shift focus to original
		saveAs("Png", datadir + condition + "/" + id + ".png");
		close();

		selectImage(image);
		close();
	
	}

	// save table with mappings
	Table.save(datadir + "ID Mappings.csv")

}
```