Posted by
John Oreopoulos on
Dec 06, 2014; 8:27pm
URL: http://imagej.273.s1.nabble.com/Is-manual-thresholding-methods-accepted-by-scientific-journals-tp5010814p5010824.html
Dear Anders,
There are two very good publications on this topic that come to mind:
Rossner, M., and Yamada, K. M. (2004). What's in a picture? The temptation of image manipulation. J. Cell Biol., 166, 11-15.
Cromey, D. (2010). Avoiding twisted pixels: Ethical guidelines for the appropriate use and manipulation of scientific digital images.
I can't find any specific comments about thresholding, but the "5th commandment" listed in Cromey's publications states:
5. Digital Images that will be Compared to one Another Should be Acquired under Identical Conditions, and any Post-acquisition Image Processing Should also be Identical:
"When images are to be compared to one another, the processing of the individual images should be identical. This includes acquisition techniques such as background subtraction or white-level balancing, which should be documented in the methods section. The same principle applies to publication figures, especially if multiple images will be published together in a single figure. This assists the reader in understanding how each image relates to the others in the group. Individual images within a figure should only be processed differently if there are compelling reasons to do so. In such cases, the differences must be explained in the methods section or the figure legend. Honesty, and completeness, are the best policies."
I think it is implied here that having an automatic / computer-guided thresholding workflow as opposed to one that requires human intervention is preferred so that all data is treated equally. But, I think as long as you are clear in your methods section about why and how you chose manual thresholding and state that you have done so, I think this would be acceptable in a paper. Ask yourself, however, would someone else have thresholded your images the same way you did? If not, they might get different results and interpretation of your analysis. Bottom line, you should supply as much information describing how you performed your image analysis especially if the crux of the paper hinges on it. Providing raw data in the supplementary materials is another way for reviewers to check the validity of your manual thresholding argument.
John Oreopoulos
On 2014-12-06, at 10:00 AM, Anders Lunde 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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