Dear Image J Users,
I am doing some work on confocal images and I am unsure as to how I should be thresholding the images before analysis.
The images consist of three channels:Blue - the axon (nerve fibre)Red - the mitochondria (subcellular structure)Green - a mitochondrial subunit
I want to determine the percentage of the axon (blue) occupied by mitochondria (red) and the the percentage deficiency of the mitochondrial subunit (green) within the mitochondria (red) - all of which I have methods for. I am unsure, however, as to how to most appropriately threshold the images.
For example:Should I be setting the same threshold for all three channels for consistency?Should I be autothresholding each channel?Should I try and manually threshold each channel based on the original images? (the problem with this being it it subjective and some of the images are hard to make out)
At the moment, I am removing all background blue channel manually then setting a high threshold (240) to find the area of the whole axon as effectively determined by eye.
I'm autothresholding the red and then setting the green channel to the same as the red. I'm worried that if I autothresholded both the computer is 'equalising' staining levels which is not helpful as we are trying to assess deficiency.
Any help would be greatly appreciated! I have tried to go into the maths and work out the best way from the algorithms but they are far beyond my skills as an undergrad biologist and I really need to finalise a method soon so I can start my analysis.
Thanks for your time!
Angus
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