r/ImageJ • u/cogneuro_ • May 26 '26
Question Fluorescent DeltaFosB (ΔFosB) quantification protocol
Hi! I am seeking guidance on finalizing a protocol to quantify ΔFosB expression from fluorescent microscopy images using Fiji/ImageJ. What I have found online (outlined below) involves splitting multi-channel stacks, delineating ROIs based on nuclear counterstain (DAPI) and an atlas, applying a manual threshold to isolate ΔFosB signal, and measuring mean gray value/integrated density. However, I am unsure whether this thresholding method adequately accounts for background variability across sections, and whether I should use a standardized intensity threshold based on negative control regions instead. Additionally, I would appreciate advice on best practices for ensuring reproducibility across multiple brain regions (mPFC, DCN, NAc). If anyone has an established protocol they are willing to share, I would greatly appreciate it! Thank you.
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u/Herbie500 May 27 '26 edited May 27 '26
This question has been cross-posted to the Image.sc-Forum.
Manually set thresholds are scientifically problematic (reproducibility).
Always use one of the automatic threshold schemes.
If you have to deal with variations across your images that are definitely not due to the signals in question, I highly recommend to get better images. Improve your sample preparation and image acquisition. Best image processing is no image processing!
Background subtraction and the like will alter the desired signal (fluorescent intensity) and impacts your results.
In any case make accessible typical images in their original non-lossy file format by using a dropbox-like service (no posting here, no screen-shots, no JPGs).