Uneven Illumination: Flatfield Correct Microscope Images

How to correct for uneven illumination across a field (brighter at one part and darker at others) when acquiring images from a microscope generally at lower magnifications (2X, 4X, 10X). This is a post-processing flatfield correction (also called Blank Field correction) when a flatfield image is available. The preferred (and faster) method is in Image J.

How to get a flatfield image with a Fluorescent-labeled Sample

You may try by using a fluorescent reference slide (Argolight,* Chroma, Valley*). Focus at brightest focal plane. Take several images by re-focusing to brightest plane each time. Make sure all images appear identically illuminated. If images vary, the flatfield image is unreliable. If you can consistently show the same uneven illumination pattern from image to image, then blur image enough to remove any artifacts via a Gaussian blur filter before using as flatfield image.
*Double check Argolight and Valley Scientific to see if these will work for near infrared dyes (e.g., CY5)

How to Flatfield or Blank Field Correct in GIMP

Same as in video, except for the following:
• Levels is found under Color > Levels
• Max and Min cannot be graphically displayed by holding down Alt key
• When pasting flatfield layer, under Edit, choose Paste As > New Layer.
• Create sampling points by holding down Control/Command key and dragging “guidelines” from ruler area. Check values in Sample Points palette (Windows > Dockable Dialogues > Sample Points)
• Click + symbol to save Levels setting.

How to Flatfield or Blank Field Correct in Image J

  1. Open unevenly illuminated image
  2. Open flatfield (shading) image
  3. Under Image > Type, select 32-bit for both images
  4. Under Process, select Image Calculator
    Enter unevenly illuminated image in the Numerator and Flatfield in the Denominator
    Select Divide
    Check Create New Window
  5. Under Image > Adjust, select Brightness/Contrast and adjust brightness to visually match unevenly illuminated image.

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