Does anybody here know how to use ImageJ to determine the OD sections of Luxol Fast Blue stained? I need aid.
I wanna compare the spare myelin in each group quantitatively using dyed spinal cord slides.
I appreciate all researchers' assistance.
Salah A. Alshehade
Open the color image in ImageJ. Split the channels - Image > Color > Split Channels. This will give you individual 8-bit images for red, green and blue. Work with the blue channel image, since Luxol Fast Blue stains myelin blue. Set the threshold (Image > Adjust > Threshold) to isolate the stained regions from background. You may need to apply some brightness/contrast adjustment first. With threshold set, analyze the image (Analyze > Measure). Make sure to check the boxes for “Integrated Density” and “Area”. The Integrated Density gives the product of Area x Mean gray value, which correlates with absorbance and OD. Measure a background region and subtract its integrated density from the sample reading to normalize. Calculate OD by using the formula: OD = log10(Integrated Density - Background)/Area
This will give you an approximate OD value that can be compared across sections. Make sure to analyze images consistently and take background readings from each image. I would suggest analyzing multiple representative regions per section to get a robust measurement.
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