I want to know if I have to keep the threshold same during analysis to compare two sets of data? for example I have a control image, when I threshold it the minimum is 30 and maximum is 255, for a treated sample it shows minimum 45 and maximum 255. Do I have to adjust the treated value to 30? or can record the default values?
I guess your question relates to 256 levels of grey scale in images. For a distribution or mixture, there is no unique threshold that will satisfy finding the edge/boundary of the particles (I'm using this term as I'm not at all sure what you're analyzing) correctly. With too much illumination the particle appears smaller; with too little then the particle appears larger. The boundary is set by the threshold and thus you would not alter the recorded values. So keep them at 30 and 45 in your case.
For something more on thresholding see this webinar:
The mathematics of image analysis
https://www.brainshark.com/malvern/vu?pi=312905118&b=1&tx=120486&c1=22
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