Thresholding a fluorescent image

[Wiggins,Jennifer M]

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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[Kenneth Sloan-2]

Why do you want a “constant threshold throughout (your) slides”?  This is almost never a good idea.

Your photos were taken using uniform imaging parameters.  This was almost (but not quite) as good as including a standard reference in each image.  In an ideal world, you need at least two known intensity levels in every image.  But, the images have been gathered - it’s too late to fix that, now.

You say you want to measure a percent area.  This indicates to me that you are *not* particularly interested in absolute intensity levels.  That’s probably good, because, while your imaging was good, it wasn’t perfect for that use.

Simple thresholding is probably NOT what you want.  There are any number of causes for absolute intensity to vary over an individual  image, and even more over a series of images.  I recommend experimenting with the (many) adaptive thresholding tools in ImageJ.  Depending on the size of the effect you see, you might consider manually editing the thresholded images.  You need to apply some judgement to evaluate how well a particular segmentation tool matches your version of the truth.

As for the bright images - before asking about how to remove it, you need a model for what caused it.  Anything you do to modify those images (absent a rock-solid model for what happened in the first place) will destroy any claims you might make about absolute intensity values.  But…if you are primarily interested in isolating a particular AREA which is different from the surround, then adaptive thresholding will handle this for you.

If you don’t mind trashing the absolute intensity levels, look at the “Adjust Brightness & Contrast” tool.  It will probably do what you want.
Using this kind of tool is morally equivalent to shooting your pictures using Auto Exposure.

If all else fails, investigate the “CLAHE” tool.

—
Kenneth Sloan
[hidden email] <mailto:[hidden email]>
Vision is the art of seeing what is invisible to others.

> On Jul 22, 2016, at 11:30, Wiggins,Jennifer M <[hidden email]> wrote:
>
> 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
>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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ImageJ mailing list: http://imagej.nih.gov/ij/list.html