Uneven Illumination By Using Image Itself for Microscope Images

How to correct uneven illumination (flatfield correct) when a flatfield image is not available from a microscope session using the image itself.

How to Correct Uneven Illumination Using Image Itself with GIMP

Follow same steps as in video, except:
• 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)
• When pasting flatfield layer, under Edit, choose Paste As > New Layer.
• Invert is found under Colors (Colors > Invert)
• Levels is found under Colors (Colors > Levels).
• Click + symbol to save Levels setting.

How to Correct for Uneven Illumination Using Image Itself with ImageJ:

Grayscale Only:

  1. Image > Duplicate
  2. Process > Filters > Gaussian
  3. Edit > Invert
  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.
  6. Image > Type, 8-bit or 16-bit.

Another Method:

Process > Subtract Background, then check Sliding Paraboloid and iteratively choose Radius until image appears evenly illuminated by eye.

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