Re: Is manual thresholding methods accepted by scientific journals?

Posted by Pål Baggethun on
URL: http://imagej.273.s1.nabble.com/Is-manual-thresholding-methods-accepted-by-scientific-journals-tp5010814p5010859.html

As long as reproducibility is fulfilled your method is compliant to scientific principles and should be acceptable to most publishers. However, as manual thresholding does not necessarily lend itself easily to reproducibility, an automated method is to be preferred.

Automatic methods, like manual ones, are subject to variability. Both accuracy and precision needs to be quantified and reported. We usually report the thresholding sensitivity, that is the change in response to a unit change in threshold, within the range of automatically or manually obtained thresholds. I find this is often neglected, rendering the results difficult, if not impossible, to interpret with any confidence.

Sincerely,
Pål Baggethun
Elkem AS, Materials Characterization Group



-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Anders Lunde
Sent: 6. desember 2014 16:00
To: [hidden email]
Subject: Is manual thresholding methods accepted by scientific journals?

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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