Thresholding a fluorescent image

Posted by Wiggins,Jennifer M on Jul 22, 2016; 4:30pm
URL: http://imagej.273.s1.nabble.com/Thresholding-a-fluorescent-image-tp5016926.html

Hello,


I am relatively new to imageJ and I am currently trying to threshold a hypoxic fluorescent stain from my tumor slides.

I am using a nitroimidazole called EF5 that binds to hypoxic cells. However, the more hypoxic, the stronger it binds. Thus this creates a gradient where the more hypoxic the area, the brighter it looks. In addition to this gradient, some images have a higher background signal (perhaps this depends on the structure or matrix composition of the tumor at that location). What I am trying to do is calculate the percent hypoxic area over the total tissue area.


My first question is, how do I establish a constant threshold throughout my slides. Can I select an area in the background and ask it to threshold at that intensity?

My second question is, what can I do with the bright images? How do I normalize them? Is there a way to align the median of all histogram plots and have a uniform background?


All the pictures from an experiment were taken under the same exposure, same magnification, on the same day.


I am open to any suggestions if there is a better way to do this.


Thank you,


Jennifer M. Wiggins
Graduate Research Assistant
Department of Radiation Oncology
University of Florida College of Medicine


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