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Hyper with Stacks A toolset facilitating manual extraction of focused slices from hyperstacks


The following toolset was designed to facilitate manual extraction of in-focus z-slices from 4D and 5D (x,y,z,t,c) sets of timelapse images such as microscopy of cancer cell migration on the chick embryo or mouse. The toolset enables a user to scroll through a 4 or 5D dataset displayed as a hyperstack, Setting the Focus (with the F1 key or by clicking the toolset button), and then split out (F2) of the hyperstack a simple 3D stack (x,y,time) of all the best focus points. Multiple channels can be split out using the index generated by setting the focus. Selecting the most focused z-position for each timepoint is possible but not necessary. The program will use the current z position for all the timepoints inbetween the previous timepoint and current timepoint. A log is printed out that can be saved as a reference.


William J Ashby and hopefully others as they make worthwhile contributions to this tool


I want to thank the creator/s of the Magic Montage toolset. Before finding Magic Montage I rarely used the toolsets or even realized that I could make them using macro language.


The toolset provides buttons to do the following:

  1. Convert a stack to hyperstack (In order to save time, the default values can be adjusted in the code).
  2. Set the focus point F1 (i.e. index your z position) for all the timepoints inbetween the previously indexed (or first) timepoint and the current timepoint.
  3. Split out the currently selected channel from your hyperstack using the index of focused z positions.
  4. Re-initialize F2, which sets the entire z index to the current z position and resets the previous timepoint to 0.


Simply save the code as a text file in the >Macros>Toolsets folder. It will show up in the toolsets dropdown under the name of the text file, Hyper with Stacks or whatever you name it. Using FIJI on a Mac this folder is found by selecting show package contents on the FIJI application.


// Hyper with Stacks.txt --version--1.5  2011-09-23
// Initial Toolset for manually extracting focused time series from hyperstacks by William J. Ashby, Vanderbilt University, Chemical and Physical Biology
// "Summary of your worthwhile addition" by "Your Name" and "Your Institute ..."
var v=versionCheck();
function versionCheck() {
    return 1;
var stackOrder = newArray("xyzct", "xyczt(default)", "xyctz", "xyztc", "xytcz","xytzc");
var displayMode = newArray("Color", "Composite", "Grayscale");
var zNum=0; var cNum=0; var tNum=0; var zz=0; var cc=0; var tt=0;
var zArray; var width;  var height;
var initialize = true;
var t0=0;
macro "Blank Action Tool - "{
macro "Stack to Hyperstack Action Tool - C000D00D01D02D03D04D05D06D07D08D09D0aD0bD0cD0dD0eD0fD10D11D12D13D15D16D1bD1cD1dD1eD1fD20D21D22D23D2bD2cD2dD2eD2fD30D31D32D33D34D38D39D3aD3bD3dD3eD3fD40D41D42D43D44D48D49D4aD4dD4eD4fD50D51D52D53D54D57D58D59D5aD5dD5eD5fD60D61D62D63D64D67D68D69D6aD6dD6eD6fD77D78D79D85D86D87D88D89D8aD8bD96D97D98D99D9aDa7Da8Da9Db0Db8CfffD14D17D18D19D1aD24D2aD3cD47D70D71D80D81D82D8fD90Da0Da1Da2Da3Da4DadDaeDafDb1Db2Db3Db4Db5DbcDbdDbeDbfDc0DccDcdDd0DdaDdcDddDe0De8De9DeaDecDedDf0Df8Df9DfaDfcDfdCf00D91D92D93Dc1Dc2Dc3Dc4Dc5Dc6Dd1Dd2Dd3Dd4Dd5Dd6Dd7De1De2De3De4De5De6De7Df1Df2Df3Df4Df5Df6Df7C00fD29D8cD9eD9fDcbDceDcfDdbDdeDdfDebDeeDefDfbDfeDffC0f0D25D26D27D28D35D36D37D45D46D4bD4cD55D56D5bD5cD65D66D6bD6cD72D73D74D75D76D7aD7bD7cD7dD7eD7fD83D84D8dD8eD94D95D9bD9cD9dDa5Da6DaaDabDacDb6Db7Db9DbaDbbDc7Dc8Dc9DcaDd8Dd9"{
// In order to make my personal defaults automatically appear in the "Stack to Hyperstack…" command, the following lines of code recreate the dialog window, display my recreation and allow user modification as necessary, and then run the "Stack to Hyperstack…" with the values provided from the dialog window.
//  Customize the defaults below when working with multiple files of data that have identical numbers of channels, z-slices, and timepoints
	Dialog.create("Convert to Hyperstack");
     	Dialog.addChoice("Order", stackOrder);  //the stackorder is found a few lines above and can be rearranged in order to put your default order first
	// To make this macro use your default number of channels, slices, and time frames
	// simply change the numbers 2, 5, and 1 in the following 3 lines of code.
	Dialog.addNumber("Channels (c):", 2);  
	Dialog.addNumber("Slices (z):", 5);  
	Dialog.addNumber("Frames (t):", 1);
	Dialog.addChoice("Display Mode:", displayMode);; 	//Displays the dialog box in order to get user input for the conversion from stack to hyperstack 
			//or hyperstack to hyperstack if you already have the data in a hyperstack 
	//Assigning the entered values to variables   
	zArray = newArray(tNum); //Initializing zArray which stores the index of in-focus z values over time
	run("8-bit");  //This line could possibly be omitted after testing for compatibility with other size stacks
	run("Stack to Hyperstack...", "order="+ans1+" channels="+cNum+" slices="+zNum+" frames="+tNum+" display="+ans2+"");
//End of dialog section
macro "F1 Set Focus Action Tool - C000C111C222D34D35D36D37D38D39D3aD3bD3cD44D48D54D58D64D68D74Da4Da5Da6Da7Da8Da9DaaDabDacC222C333C444C555C666C777C888C999CaaaCbbbCcccCdddCeeeCfffD00D01D02D03D04D05D06D07D08D09D0aD0bD0cD0dD0eD0fD10D11D12D13D14D15D16D17D18D19D1aD1bD1cD1dD1eD1fD20D21D22D23D24D25D26D27D28D29D2aD2bD2cD2dD2eD2fD30D31D32D33D3dD3eD3fD40D41D42D43D45D46D47D49D4aD4bD4cD4dD4eD4fD50D51D52D53D55D56D57D59D5aD5bD5cD5dD5eD5fD60D61D62D63D65D66D67D69D6aD6bD6cD6dD6eD6fD70D71D72D73D75D76D77D78D79D7aD7bD7cD7dD7eD7fD80D81D82D83D84D85D86D87D88D89D8aD8bD8cD8dD8eD8fD90D91D92D93D94D95D96D97D98D99D9aD9bD9cD9dD9eD9fDa0Da1Da2Da3DadDaeDafDb0Db1Db2Db3Db4Db5Db6Db7Db8Db9DbaDbbDbcDbdDbeDbfDc0Dc1Dc2Dc3Dc4Dc5Dc6Dc7Dc8Dc9DcaDcbDccDcdDceDcfDd0Dd1Dd2Dd3Dd4Dd5Dd6Dd7Dd8Dd9DdaDdbDdcDddDdeDdfDe0De1De2De3De4De5De6De7De8De9DeaDebDecDedDeeDefDf0Df1Df2Df3Df4Df5Df6Df7Df8Df9DfaDfbDfcDfdDfeDff"{
	run("Set Focus [F1]");
macro "Set Focus [F1]" {
		if (zArray==0){
			if (Stack.isHyperstack) {
				Stack.getDimensions(width, height, cNum, zNum, tNum);
			} else {
				print("Close the error window and click the Run Stack to Hyperstack button");
		Array.fill(zArray, zz);
	for (i=t0; i<tt; i++){
	if (t0<tt) 
		print("z is set to: "+zz+"  for t: "+t0+" through "+tt);
	if (t0>=tt) 
		print("current t="+tt+"  Proceed to t>"+t0+" or re-initialize");
macro "Split Stack Action Tool - R9077C888R9977R0977"{
    if (nSlices==1)
      		exit("Stack required");
    stack1 = getImageID;
    w = getWidth; h = getHeight;  title = getTitle;
    stack2 = 0;
    n = zArray.length+1;
    for (i=1; i<n; i++) {
        showProgress(i, n);
        if (stack2==0) {
            newImage("Focused_C"+c+"_"+title, "8-bit", w, h, 1);
            stack2 = getImageID;
        } else {
            run("Add Slice");
    print("Successfully split out channel "+c+"\nTo split out a different channel using this z-index, \nsimply change the channel in the hyperstack and repeat"); 
macro "F2 ReInitialize Action Tool - C000C111C222D14D15D16D17D18D19D1aD1bD1cD24D28D34D38D44D48D54D85D86D8bD8cD94D9aD9cDa4Da9DacDb4Db8DbcDc5Dc6Dc7DccC222C333C444C555C666C777C888C999CaaaCbbbCcccCdddCeeeCfffD00D01D02D03D04D05D06D07D08D09D0aD0bD0cD0dD0eD0fD10D11D12D13D1dD1eD1fD20D21D22D23D25D26D27D29D2aD2bD2cD2dD2eD2fD30D31D32D33D35D36D37D39D3aD3bD3cD3dD3eD3fD40D41D42D43D45D46D47D49D4aD4bD4cD4dD4eD4fD50D51D52D53D55D56D57D58D59D5aD5bD5cD5dD5eD5fD60D61D62D63D64D65D66D67D68D69D6aD6bD6cD6dD6eD6fD70D71D72D73D74D75D76D77D78D79D7aD7bD7cD7dD7eD7fD80D81D82D83D84D87D88D89D8aD8dD8eD8fD90D91D92D93D95D96D97D98D99D9bD9dD9eD9fDa0Da1Da2Da3Da5Da6Da7Da8DaaDabDadDaeDafDb0Db1Db2Db3Db5Db6Db7Db9DbaDbbDbdDbeDbfDc0Dc1Dc2Dc3Dc4Dc8Dc9DcaDcbDcdDceDcfDd0Dd1Dd2Dd3Dd4Dd5Dd6Dd7Dd8Dd9DdaDdbDdcDddDdeDdfDe0De1De2De3De4De5De6De7De8De9DeaDebDecDedDeeDefDf0Df1Df2Df3Df4Df5Df6Df7Df8Df9DfaDfbDfcDfdDfeDff"{
	run("Re-Initialize [F2]");
macro "Re-Initialize [F2]" {
	print("Re-initialized, proceed with selection of focused z slices");
var pmCmds = newMenu("Popup Menu",
        newArray("Set Focus [F1]", "Re-Initialize [F2]","-","Copy", "Paste","Rename...")
macro "Popup Menu" {
    cmd = getArgument;


The above code is made freely available for use and modification in keeping with the Creative Commons license see CC Attribution-Noncommercial-Share Alike. ImageJ/FIJI programmers and aspiring programmers are welcome to contribute here. I, William J Ashby, the initial creator and first author invite others who make worthwhile contributions to add themselves to the list of authors both on this wikipage and in the above code.

Known Bugs

The program will throw an error if you start setting focus points (i.e. indexing z positions) on a stack without converting it to a hyperstack. A message to run Stacks to Hyperstacks is displayed and the error doesn't cause problems otherwise in the program.

Future Additions/Improvements

Anyone and everyone are welcome to take what I have started and implement new buttons and features. You may also request additions to the toolset here but I will probably have to leave the development of future additions to others.

  1. The ability to write the index of in-focus z positions to file and read in the index from this file.
plugin/stacks/toolset_for_manual_extraction_of_best-focused_z-slices_from_hyperstack/start.txt · Last modified: 2019/04/12 13:13 by

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