Hello everyone, I am seeking guidance on the analysis of scanned films obtained from Proteome Profiler Mouse Cytokine Array Kits, specifically focusing on the quantification of dot intensity using ImageJ. I have successfully scanned the array membranes, which contain dots exhibiting varying intensity levels, ranging from intense to very light. However, I am facing challenges in determining the optimal method for selecting the appropriate area for intensity measurement, particularly due to the variability in dot intensity across the membranes.
I am uncertain about the most suitable approach for measuring dot intensity and comparing them across the membranes. Considering the diverse range of dot intensities present on the membranes, I am unsure whether to fix the area for intensity measurement or adopt a different approach.
I would greatly appreciate guidance or recommendations on how to effectively analyse the scanned films to quantify dot intensity using any available protocol or instruction using ImageJ. Specifically, I am seeking insights on selecting the appropriate area for intensity measurement, taking into account the presence of both intense and light dots.
I have attached a sample image of the scanned film for reference, and I am more than willing to provide additional details or clarification upon request.
Hello, you should use the area of the largest dot as the reference for other dots and measure mean or median intensity sustracted by a measure of your film background
The three pairs of dots in the corners are your reference controls. You should normalize the intensity of the other pairs to those.
In regards to ROI selection, every dot should have the same size in this blot. The control dots are overexposed and have bled into adjacent pixels, so I wouldn't just use the size of the largest dot for your measurements. The pair of dots near the middle of the top image looks to be the proper size, so I would recommend those as the "ideal" area. However, you can also use the integrated signal density (equivalent to the mean intensity * area) to deal with differently sized ROIs.
I recommend using the Licor Image Studio (https://www.licor.com/bio/image-studio/, the Lite version is free to use) for densitometric quantification. They make it simple to do local background subtraction and make it easier to assess the shoulders of your ROI.
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