Tips from the blog VII: Recolour Z-stack and Save Projection

I’m putting this up here in case it is useful for somebody.

We capture Z-stacks on a Perkin Elmer Spinning Disk microscope system. I wanted to turn each stack into a single image so that we could quickly compare them. This simple macro does the job.

  1. We import the images straight from the *.mvd2 library using the wonderful BioFormats import tool. We open all files as composite hyperstacks.
  2. This Macro is then run (works on all of the open images).
  3. It finds the mid-Z point of the stack and sets the brightness/contrast for each channel to Auto.
  4. It then recolours the channels to the right colours (we capture DAPI then GFP then mCherry, but the import colours them red, green and blue, respectively).
  5. Then it makes a z-projection and saves the file in a directory that you specify at the start.
  6. It closes the projection and the z-stack.

You can then open up all of the projections, tile them and take a look at what was going on in the experiment.


setBatchMode(true);
imgArray = newArray(nImages);
dir1 = getDirectory("Choose Destination Directory ");
for (i=0; i<nImages; i++) {
	selectImage(i+1);
	imgArray[i] = getImageID();
	}
for (i=0; i< imgArray.length; i++) {
	selectImage(imgArray[i]);
	id = getImageID();
	win = getTitle();
	getDimensions(w, h, c, nFrames, dummy);
	Stack.setSlice(round(nFrames/2));
	run("Enhance Contrast", "saturated=0.35");
	run("Next Slice [>]");
	run("Enhance Contrast", "saturated=0.35");
	run("Next Slice [>]");
	run("Enhance Contrast", "saturated=0.35");
	Stack.setChannel(1)
	run("Blue");
	Stack.setChannel(2)
	run("Green");
	Stack.setChannel(3)
	run("Red");
	run("Z Project...", "projection=[Max Intensity]");
	saveAs("TIFF", dir1+win);
	close();
	selectImage(id);
	close();
	}

Sorry for lack of commenting.

Tips from the blog is a series and gets its name from a track from Black Sunday by Cypress Hill.

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