phf1-extraction.Rmd

---
title: "Tangle Channel Extraction"
description: |
 This ImageJ script extracts the PHF1 and DAPI channels from multi-channel TIFF images, merges both channels, and assigns each image a random alphanumeric code for blinded tangle annotation and classification as intraneuronal vs. extraneuronal.
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 "PHF1 Channel Extraction" {

	setBatchMode(true);

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

	dir = "<insert your directory here>";
	datadir = dir + "Data/2 - Channel Extraction/Tangle/";


	////////////////////////////////////////////////////////////
	/////  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 PHF1 AND DAPI CHANNELS
		////////////////////////////////////////////////////////////
		
		// duplicate PHF1 channel and merge with HuC/D
		selectImage(image); // shift focus to original
		run("Duplicate...", "title=PHF1 duplicate channels=17");
		selectImage(image);
		run("Duplicate...", "title=DAPI duplicate channels=1");

		// merge channels
		run("Merge Channels...", "c2=PHF1 c3=DAPI create");
		
		selectWindow("Composite"); // shift focus to original
		saveAs("Png", datadir + condition + "/" + id + ".png");
		close();

		selectImage(image);
		close();
	
	}

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

}
```