Tag Archives: microscopy

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.

Green is the Colour: mNeonGreen spectra

I was searching for the excitation and emission spectra for mNeonGreen. I was able to find an image, but no values for the spectra. Here is an approximation of the spectra (xlsx format, still haven’t figured out csv for wordpress).

I got these values using IgorThief.ipf a very handy tool that allows the extraction of XY coordinates from a bitmapped plot (below).

mNeonGreen is available from Allele Biotechnologies.

Here is a great site for comparing fluorescent protein properties.

Edit @ 06:54 4/7/14: A web-based data thief was suggested by @dozenoaks

The post title is taken from “Green is the Colour” from the Pink Floyd LP “More”