Help with quantifying pictures after channel splitting

Posted by Stephanie Klett on
URL: http://imagej.273.s1.nabble.com/Help-with-quantifying-pictures-after-channel-splitting-tp5011445.html

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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