Uneven Illumination Correction for Phase DIC EM Microscope Images

How to correct for uneven illumination (dark and lighter areas on an image) when images are from EM microscopes, when phase contrast methods are used, and when Differential Contrast Interference (DIC) methods are used.

How to Correct DIC, EM, Phase Images for Uneven Illumination in GIMP

Create a duplicate layer of the original above original image (Layer>Duplicate 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)

Filter > Enhance > High Pass
Set High Pass filter Standard Deviation so that dark and light spots of image are within 5 points or so on Info palette readout for sampler points.
Radius is generally high enough so that details are not lost, and so that uneven illumination is largely corrected.

Save the Preset by clicking + symbol.

Restore contrast: Under Colors select Levels: Click Logarithmic icon (far right at top) and then match black level slider and white level slider to ends of the histogram.

How to Correct for Uneven Illumination for DIC, EM and Phase in Image J

  1. Under Edit > Options > Appearance choose correct bit depth to match image (bit depth can be found under Image > Show Info).
  2. Under Process select Subtract Background.
    In Subtract Background go with Rolling Ball radius value of 50. Check Light Background (brightfield only), Disable Smoothing and Preview.
    Visually determine result and iteratively set Rolling Ball radius until value that works by eye is found.
    Not all images work with this method: colors can get skewed, and no means are available to track uniformity of tones.

See the Instruction Page for an index of all instructional videos and documents
See Image Ethics for image integrity information on how to avoid scientific misconduct

Leave a Reply