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What is flatfielding?

Flatfielding is a procedure that will allow you to correct images for uneven illumination. Even with well-maintained objectives and good samples, illumination over a large field of view will always be uneven.

Why should you flatfield your color images?

To compensate for the fact that the edges of the field of view are dimmer than the center, due to uneven illumination. After flatfielding your images will look better (see below).

What do you need to do this?

You will need to acquire a blank image, and you will need to use one of MSL’s flatfielding macros.

What results can you expect?

Here is an example of an image before flatfielding, the blank image used for the flatfielding procedure, and the flatfielded result:

How do you acquire a blank image?

  1. Select the proper objective you intend to use.
  2. Make sure the TL lens is in the correct position (out for 5X and lower magnification objectives, in for the rest).
  3. Focus on your sample.
  4. Autoexpose
  5. Remove the sample slide, autoexpose again.
  6. Apply white balance.
  7. Make sure the image without the slide is not saturated.
  8. Acquire an image without the slide; this will be your blank image for that objective.

What do you do after acquiring the blank image?

  1. Put your slide back on.
  2. Take all other images without changing the settings.

How should you save your images (in Volocity software)?

  1. As TIFFs (make sure you click on “Options…” and ensure “Convert to RGB for publication” is clicked and “Scale” is unclicked, every time you save a batch of images).
  2. Groups of images taken with different objectives and/or settings should be placed in different folders.

Important things to keep in mind when acquiring a blank image:

  1. You will need a different blank image for every objective you use, if you use more than one objective.
  2. You will need a different blank image if you use different settings.
  3. Blank images are best fresh, like sushi (thanks to Jim Sims at Hamamatsu for that analogy); you will need new blank images every time you come to MSL.

How do you flatfield your images with the MSL macro?

The macro will ask you for the objective you used, the flatfield image, and the directory with the images that needed to be flatfielded. Within that directory, it will create a new folder called Flatfielded_Images, and place the flatfielded images in that folder.

How do you install the MSL flatfielding macro?

  1. Download and install FIJI.
  2. Download the macros and unzip them.
  3. Make a folder called MSL in the plugins folder in the FIJI folder. (To get inside this folder on a mac, go to wherever FIJI is—typically in Applications—right-click on it and select Show package contents). You will now see the folders inside FIJI, one of which is plugins.
  4. Put the following files inside FIJI/plugins/MSL:
    1. _FlatBF_BX61v2
    2. _FlatBF_IX81v2
  5. Restart FIJI