Analysis of multiple different image sets by fluorescence intensity - how to ensure consistency across different image sets

I am trying to create a pipeline that is consistent for my propidium iodide (red-- a marker for cell death) and beta-III-tubulin (green-- a marker for neuronal cells) immunostaining. My end goal is to have a percentage of dead neurons for treated and untreated groups to see if treatment helps prevent cell death.

I run an experiment every two weeks and grow enteric neurons from mice, so they are a primary cell culture and therefore are less consistent and reliable than say a cancer cell line. I am classifying whether or not a cell is 1) Neuronal 2) Dead and 3) Neuronal and Dead. So at the end I get a percentage of the fraction of dead neurons over total neurons.

The problem is I set my intensity thresholds at a set value and was intending to keep these thresholds the same (as well as the exposure time, etc.) so that I could ensure that all of the images are analyzed in the same way. However, this week my neurons weren’t as bright with the same exposure time etc. and all of them are under the intensity thresholds. I don’t want to change my thresholds between experiments as a control. Is there a way to normalize the intensity such that I would still be able to compare between groups? I saw there is a RescaleIntensity function but I don’t know how it works.

Any assistance would be GREATLY appreciated! :slight_smile:


That can be the problem with manual thresholds- they rely on getting the exact same staining every time, and biology and samples are rarely so accommodating!

You could use RescaleIntensity, but that has its issues as well- my recommendation would be to instead find a thresholding method like Otsu, etc that works well on your images and use that instead -you can still set reasonable upper and lower bounds for the threshold if you want to keep the threshold within a range.