http://imagej.273.s1.nabble.com/Is-manual-thresholding-methods-accepted-by-scientific-journals-tp5010814p5010822.html
In my experience, it is worth the time to find the best auto-threshold for your data set, and then stick to it. Of course, which method is “the best” varies with the images being analyzed. You just have to test them, do a few trial analyses, and find one that produces reasonable numbers that reflect what you see qualitatively. I agree with your advisor. It is not worth the risk using manual eyeball methods and then having a reviewer raise a problem with bias, whether it be real or perceived.
If you are analyzing a Z-stack, be sure you move to a bright image in the middle of the stack before applying the threshold to all images in the stack. If you apply a threshold to the first image in the stack and it has almost no signal, the thresholding will not be optimal.
A recent example of how we applied Triangle successfully for colocalization analysis can be found in Traffic 15:1344 (2014). This method was robust and the numbers made sense with respect to the biology. For some of the data, which were acquired using a different method, Triangle did not work well, but Otsu did. Your mileage may vary.
> On Dec 6, 2014, at 10:00 AM, Anders Lunde <
[hidden email]> wrote:
>
> Dear mailing list,
>
> I have developed a nice macro for identifying colocalized signals for
> z-stack confocal images with multiple channels/colors. However, my
> advisor/professor has now come to question my method for setting a
> threshold for signal/no-signal in the infividual channels.
>
> My manual method has been to simply raise the threshold above what I
> relatively confidently can see is background, like large areas with no
> apparent staining. The reason I did it manually is because when I played
> around with the automatic thresholding methods in ImageJ I decided that
> they were not any better than manual and could be subject to mistakes.
>
> My supervisor now feels that this sounds too subjective and would not look
> good in a paper. He therefore asked me to try to find a way that was more
> guided e.g. by the histogram or something, anything that is less subjective
> (not sure if he is worried about accuracy or how it sounds in a paper).
>
> What is the current standard for this kind of analysis in scientific
> journals, in particular with regards to the acceptability of manual
> thresholding of immunofluorescent brain sections stained with various
> antibodies (and nuclear markers and neuron trancers)? Is there a preference
> for automated, manual or some hybrid methods? Could I "get-away" with
> something like this: "Thresholds were set manually at a level that
> excluded most pixels in assumed background areas. Inspection of the
> assigned threshold level in the ImageJ intensity histogram showed that the
> thresholds were set at where the main peak (background pixels) started to
> or had reached a minimum value."
>
> Image set that Im working on:
>
> I am working with images of brain sections with 4 colors/channels: nuclear
> stain, two immunofluorescence staining for transciption factors (nuclear
> localization), and a retrograde nerve cell staning (nuclear + cytoplasm
> staining).
>
> Greateful for any advice!
>
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