Can anyone please advice on optimization of comet images (contrast , gamma settings). What range of gray scales will be suitable?
I suggest tagging this question with "image processing" or "digital image processing", it may help experts in those areas (the ones that will most likely) have an answer for you.
Be precise. What exactly are you optimizing? So it is easier to see? Trying to determine what gamma level to see the image (automatically) for that the gray scales are easier to look at for the image visually? These may help experts that may be able to answer your question.
thank you very much for your advices . I have attached here my healthy DNA /comet images . Kindly let me know things that are wrong .
I had to google how comet assay images are supposed to look. It seems that the black holes in the center and the rings should not be there, and that there should be comet-like tails.
It could be that the image you posted suffers from overflow, which happened when converting the raw data to 8 bits jpeg. This image is beyond repair. So I ask again: could you post the RAW data?
i know , even i have doubts on the image . The cells are human lymphocytes . May i also know which cell you worked on Parisa ??
Dear Rinchen, I also did comet assay using human and rat lymphocyte. I isolated the lymphocyte using ficoll gradient paque and stained it with Vista Green DNA dye. The result I got is like picture below. I'm not sure what cause the halo and black hole in the center of your nucleoids. Is it because the lysis process is incomplete? It looks like a membrane (I'm not sure)? In order to get the comet we need to lysis the cell membrane and also the nucleus mebrane, so the DNA can be exposed and pushed by electrical current. So make sure you give enough lysis time. I put my agarose for 1 hour in lysis buffer, some source mentioned It can be put overnight in lysis buffer to increase the sensitivity.
This is my first time doing comet assay and also first time this method done in my center. I would be happy if anyone share their result and method, because I still optimized my method. Thank You
Regards,
Raesa
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