reorder-channels.Rmd

---
title: "Reorder Channels"
description: |
 This ImageJ script allows the user to reorder imaging channels as necessary to facilitate downstream analysis.
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 "Reorder Channels [r]" {

    // setBatchMode(true); // will bypass GUI!

    dir = "<insert your directory here>";
    output = dir + "Converted TIFF Crops/Reordered Crops/";

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

    
    for (f = 0; f < files.length; f++) {

        open(input + files[f]);
        
        ////////////////////////////////////////////////////////////
        /////  LOAD IMAGE + DEFINE MARKERS
        ////////////////////////////////////////////////////////////
        
        image = getTitle(); // get crop title
        selectImage(image); // shift focus to the selected crop
        filename = substring(image, 0, indexOf(image, ".tif"));
        
        run("8-bit"); // convert to 8-bit, note that will lose fluorescence granularity
    
        // create array to hold assigned slice names
        titles = newArray(nSlices);
        // list possible marker names
        markerNames = newArray("GFAP", "DAPI", "MHC2", "TSPO", "EAAT2", "TMEM119", "CD68", "EAAT1", "ALDH1L1", "IBA1", "Vimentin", "Ferritin", "HuCD", "YKL40", "GS", "Abeta", "PHF1-tau");
        
        for (i = 1; i <= nSlices; i++) {
             
            setSlice(i);
            run("Enhance Contrast...", "saturated=0.3"); // only for visualization purposes
    
            // call dialog box to assign marker name to selected slice
            Dialog.create("Which Marker Is This?");
            Dialog.addChoice("Type:", markerNames);
            Dialog.show();
            marker = Dialog.getChoice();
    
            // set slice name based on user choice
            setMetadata("Label", marker);
            titles[i-1] = getInfo("slice.label");
            
            markerNames = Array.deleteValue(markerNames, marker); // prevents the same marker from being assigned to multiple slices
            
        }   
    
        ////////////////////////////////////////////////////////////
        /////  REORDER SLICES + DEFINE LUT
        ////////////////////////////////////////////////////////////
        
        run("Stack to Images"); // separate each slice
    
        // concatenate in right order - rearranges all slices
        run("Concatenate...", " title=" + image + " open image1=DAPI image2=ALDH1L1 image3=IBA1 image4=GFAP image5=MHC2 image6=TSPO image7=EAAT2 image8=TMEM119 image9=CD68 image10=EAAT1 image11=Vimentin image12=Ferritin image13=YKL40 image14=GS image15=HuCD image16=Abeta image17=PHF1-tau");
    
        // make composite image with color
        run("Make Composite", "display=Color");
        
        finalNames = newArray("DAPI", "ALDH1L1", "IBA1", "GFAP", "MHC2", "TSPO", "EAAT2", "TMEM119", "CD68", "EAAT1", "Vimentin", "Ferritin", "YKL40", "GS", "HuC/D", "Abeta", "PHF1-tau");
        colorList = newArray("Blue", "Red", "Green", "Magenta", "Cyan", "Yellow", "Grays");
                
        for (k = 1; k <= nSlices; k++) {
             
            setSlice(k);
            setMetadata("Label", finalNames[k-1]); // set final name of slice
    
            run(colorList[(k-1) % colorList.length]); // apply distinct false color per slice
            
            // add more data pre-processing if desired!
            
        }
        
        saveAs("Tiff", output + filename + "_Reordered.tif");
        
        close();

    }
    
    
}
```