Posted by
MrScientistman2 on
Dec 06, 2014; 3:00pm
URL: http://imagej.273.s1.nabble.com/Is-manual-thresholding-methods-accepted-by-scientific-journals-tp5010814.html
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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