We’re gonna need a montage!

While lots of image analysis and processing is about getting quantifiable numbers out of imaging data, inevitably, there comes a point when you just need to show off some pretty pictures in a presentation, poster or blog (who knew?).

Who would have thought autofluorescence would be so pretty?

Who would have thought autofluorescence would be so pretty?

In this post we’ll be looking at a couple of ways to display your oh-so pretty multichannel pictures.

Ways to Display

Depending on how you open your multichannel image in Fiji, what you see will vary. Using the Bioformats importer, I see the following:

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Note the navigation bar at the bottom of the image window. This lets you move between slices in a stack (be they channels, timepoints or z-slices). Multichannel images in Fiji can be displayed in one of three ways:

  • Greyscale: show each channel as greyscale
  • Color: show each channel with a pseudocolour lookup table (colours may be saved from the acquisition system)
  • Composite: Use the colour information but overlay the channels.
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Displaying a multichannel image in (L to R) Greyscale, Color and Composite mode.

Importantly, it’s possible to view and change the display mode using the Channels tool [Image > Color > Channels Tool].

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You can access a bunch of handy commands from the More button but also (in composite mode) turn individual channels off and on using the check boxes.

Getting the Image Out

If you’ve turned off any channels you don’t want, you can either save your image [File > Save As > PNG], or copy the image and paste it into your program of choice. The image is copied at full resolution but with only the active channels included.

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Example of pasting data into Inkscape. The left image was copied with all channels active while the right image had two deselected before copying.

NOTE: For some reason (probably to do with speed), Fiji uses two clipboards for copy/paste actions. In order to copy data out of Fiji into other things, it’s important to use [Edit > Copy to System] instead of the default [Edit > Copy].

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Even Rocky Had a Montage…

Sometimes, it’s useful to create a montage of your channels in combination with a composite. Something like this perhaps?

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It’s probably a lot easier than you think. Here’s how:

      1. Split the multichannel image using [Image > Split Channels]
      2. Set each of the channels to use a “Grays” lookup table [Image > Lookup Tables > Grays]. This is also available on the Channels Tool “More” button (told you it was useful)
      3. If you want a text label (as above) use [Image > Rename] to set the name of the images (eg. “Red”, “Blue” or whatever)
      4. Merge the channels back into a stack using [Image > Color > Merge Channels] selecting the right colours for each image. Make sure to select “Create Composite” and “Keep Originals”.
      5. On the resulting composite, run [Image > Type > RGB color]. Repeat step 3 to label the composite (“Merge” is a good choice)
      6. Close the 3-channel composite (just keep the RGB stack open). You should now have the four gray channels plus one composite.
      7. Put all of the images together into a stack using [Image > Stacks > Images to Stack]. Check the box to use Titles as labels and hit OK.
      8. The last step is to turn the stack of images into a montage using [Image > Stacks > Make Montage]. The options are fairly self-explanatory. Hit OK

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If you’ve chosen the default layout and checked the “Label Slices” box you’ll have a 3×2 grid with one blank frame (I like putting a border of 2-5 pixels between images):

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If you’d prefer a single row, you can always specify the dimensions as 5 columns x 1 row which looks like this:

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Epilogue

This post has been dealing with multichannel data but there are plenty of circumstances where you may want to use montages to display other multidimensional data. Below is a montage of a time series, but you could just as easily use this method to display a z-stack.

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