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:
- Image > Duplicate
- Process > Filters > Gaussian
- Edit > Invert
- Under Process, select Image Calculator
Enter unevenly illuminated image in the Numerator and Flatfield in the Denominator
Check Create New Window
- Under Image > Adjust, select Brightness/Contrast and adjust brightness to visually match
unevenly illuminated image.
- Image > Type, 8-bit or 16-bit.
Process > Subtract Background, then check Sliding Paraboloid and iteratively choose Radius until image appears evenly illuminated by eye.