Posted by
Stoyan Pavlov on
URL: http://imagej.273.s1.nabble.com/quantify-the-fibers-with-the-red-color-tp5003734p5003750.html
Hi Thuraya
In brightfield images one SHOULD NOT quantify brightness but density,
because the image is formed via absorbtion of certain quantity of light of
certain wavelength.It is a very common mistake to treat brightfield images
like fluorescence images. In the brightfield image the light passes through
the specimen and is absorbed by the different dyes. So the dye that appears
red would have absorbed mostly blue and green light, this means that the
red dye will be densest (in greatest quantity) at the places where there is
no blue or green light passing at all , this is wherever the blue or green
pixels are the darkest.
In your case the "meaningful" channel would be either BLUE or GREEN but NOT
RED. This is so, because the red appearing fibers would have absorbed this
colors and left red light pass true - that is why the red channel will be
less informative (in this channel you are actually quantifying the green or
blue stain which absorbed the red light).
That is why you will need to do the thresholding in one of these other
channels (chose the one in which the fibers seem denser (darker)). Next in
Analyze->Select measurements you should select Area and Area fraction with
the Limit to Threshold option checked. Then you run Analyze->Measure
(Ctrl+M) and you will receive total thresholded area in pixels and
Thresholded area fraction in %.
Another aproach would be to Invert the image and than quantify the
brightness in the Red channel of the Negative.
Stoyan
2013/7/3 Benjamin Grant <
[hidden email]>
> Running "subtract background" after threshholding will not do anything. Try
> running that first, then thresholding, that might be your issue.
>
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Dr. Stoyan P. Pavlov, MD, PhD
Departament of Anatomy, Histology and Embryology
Medical University "Prof. Dr. Paraskev Stoyanov", Varna
Prof. Marin Drinov Str.55
9002 Varna
Bulgaria
Tel: +359 (0) 52 - 677 - 052
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