Posted by
G. Esteban Fernandez on
URL: http://imagej.273.s1.nabble.com/Help-with-quantifying-pictures-after-channel-splitting-tp5011445p5011507.html
Hi Stephanie,
For this kind of thing the Colour Deconvolution plugin usually works well
for me (if staining is consistent from image to image). The plugin would
separate your color images into two images corresponding to the two
histological stains you used.
In addition to Colour Deconvolution I'd apply a Process > Subtract
Background step (right now I don't remember if background subtraction works
better before or after color deconvolution).
You could also try the Auto Local Thresholding plugin instead of the global
thresholding you described.
In short, my workflow is generally this:
1. Colour Deconvolution (comes with Fiji ImageJ)
2. Process > Subtract Background
3. Auto Local Threshold (also in Fiji)
-Esteban
On Feb 4, 2015 6:58 AM, "Stephanie Klett" <
[hidden email]>
wrote:
> Dear Image J discussion group,
> I have a question about how I can quantify my digital microscopic images
> (.jpg) In my case I done a immunological staining where red is the colour
> of
> interest. Therefore, I split the channels and use the blue labelled picture
> and apply the function Threshold... (under Image-->Adjust-->). Then I get a
> histogram and an automatically adjustment of the colour sensibility (?). My
> question is what should I keep in mind when I want to compare several
> images. Do I have to adjust always the same sensibility (from the rage of 0
> to 255) or do I choose the automatically adjustment? My problem is, that
> sometimes the automatically adjustment represent the quantification better
> than the sensibility rate that I decided to use and other way around. Which
> way is more unbiased or do I have to do a completely other procedure?
>
> Here I display screen shots where I document my way and may problem more
> exemplified.
>
> Fig. 1 Opened a file and split the channels
>
> Fig. 2 Apply the function ³Threshold...² on the image labelled "blue³
>
> Fig. 3 Display the automatically adjustment of 228
>
> Fig. 4 Corrected the threshold to 180
>
> Fig. 5 A new image, display the automatically threshold of 222
>
> Fig. 6 Corrected the threshold to the undoing rate of 180
>
>
> I¹m very happy if you can help me and looking forward to your answer.
>
> Thank you for your attention
>
> Best regards
> Stephanie
>
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