Microscope Images: Tonal Adjustment For Densitometry, Linear Histogram Matching

How to adjust tones to a standard brightness level when images are taken over several microscopy sessions. Images are assumed to be grayscale.

How to Adjust Tones for Densitometry in GIMP

Follow steps shown in video. Show Histogram by choosing Histogram in Windows > Dockable Dialogs. Levels is located under Colors > Levels.

How to Adjust Tones for Densitometry in Image J

  1. Open images and show histogram for reference image (image to which others are being made to match): Analyze > Histogram.
  2. Use cursor to find background value (read from Image J bar and write down: this is the reference value). The background would be the non-specimen area. It’s also assumed the image is evenly illuminated. Cursor reads only single pixels: better to make a rectangle with the marquee tool and close/open histogram to see mean value from rectangle area.
  3. Open images you need to match, one by one. Open Brightness Contrast window: Image > Adjust > Brightness/Contrast.
    Move Maximum slider by eye and measure background value until it matches reference value. You will need to measure from several points or make a rectangle with the marquee tool and close/open histogram to see mean value from rectangle area.
    Fine tune by matching background value to reference image visually in the event that the values don’t match up backgrounds visually. It’s assumed you set the Appearance correctly: Under Edit > Options > Appearance choose correct bit depth to match image (bit depth can be found under Image > Show Info).

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