What is subtracted in 'Image subtraction'?

[-may]

Hello ImageJ users,I am trying to compare two images before and after an enzyme treatment. You will see some features disappeared in the 'after' image.  To show thedifference, I tried to subtract the 'before' image using the 'after' one. However, it does not seem to make sense of what I got.I am wondering how the image was subtracted, based on what number?The images could be seen athttp://www.flickr.com/photos/youngkyi/8532520348/in/photostreamhttp://www.flickr.com/photos/youngkyi/8531410143/in/photostreamhttp://www.flickr.com/photos/youngkyi/8531410161/in/photostreamThank you very much!Tian    

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[Michael Schmid]

Hi Tian,

if you subtract two images, you may get positive or negative values.
Select '32-bit (float) result' to see also the negative ones; otherwise only the positive values will be shown, and the negative ones will become 0.

Your input images have different average brightness, that's why it seems that your subtraction has resulted in mostly black pixels (zero, because non-float images can't have negative values).

Michael
________________________________________________________________
On Mar 5, 2013, at 22:00, -may wrote:

> Hello ImageJ users,I am trying to compare two images before and after an enzyme treatment. You will see some features disappeared in the 'after' image.  To show thedifference, I tried to subtract the 'before' image using the 'after' one. However, it does not seem to make sense of what I got.I am wondering how the image was subtracted, based on what number?The images could be seen at
> http://www.flickr.com/photos/youngkyi/8532520348/in/photostream
> http://www.flickr.com/photos/youngkyi/8531410143/in/photostream
> http://www.flickr.com/photos/youngkyi/8531410161/in/photostream
> Thank you very much!Tian    
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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[Cammer, Michael]

This may help to see the differences between the two images.
http://www.flickr.com/photos/mcammer/8532618454/
Magenta shows features that are only in the before image.  Cyan shows features only in after.  Yellow shows features common to both images.
This was done using the color merge function where red is before, green is after, and blue is the calculated difference between before and after.  It could have been done only merging the before and after in red green too.

To answer your question regarding how the image was subtracted:  Each XY location in each image has a discrete value from 0 (black) to 255 (white).  Process > Image Calculator operates on each XY location in Image1 and Image2 to give a resultant image.  Because you are using 8 bit math, negative values are clipped to zero.

If you subtract after from before, then pixels at each location that are brighter in after end up zero. Here is an example where magenta is before minus after and cyan is after minus before.  If these color channels were separated, the you would see that the blue channel is the difference of before and after.
http://www.flickr.com/photos/mcammer/8531541063/


________________________________________________________
Michael Cammer, Assistant Research Scientist Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of -may
Sent: Tuesday, March 05, 2013 4:01 PM
To: [hidden email]
Subject: What is subtracted in 'Image subtraction'?

Hello ImageJ users,I am trying to compare two images before and after an enzyme treatment. You will see some features disappeared in the 'after' image.  To show thedifference, I tried to subtract the 'before' image using the 'after' one. However, it does not seem to make sense of what I got.I am wondering how the image was subtracted, based on what number?The images could be seen athttp://www.flickr.com/photos/youngkyi/8532520348/in/photostreamhttp://www.flickr.com/photos/youngkyi/8531410143/in/photostreamhttp://www.flickr.com/photos/youngkyi/8531410161/in/photostreamThank you very much!Tian    

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-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of -may
Sent: Tuesday, March 05, 2013 4:01 PM
To: [hidden email]
Subject: What is subtracted in 'Image subtraction'?

Hello ImageJ users,I am trying to compare two images before and after an enzyme treatment. You will see some features disappeared in the 'after' image.  To show thedifference, I tried to subtract the 'before' image using the 'after' one. However, it does not seem to make sense of what I got.I am wondering how the image was subtracted, based on what number?The images could be seen athttp://www.flickr.com/photos/youngkyi/8532520348/in/photostreamhttp://www.flickr.com/photos/youngkyi/8531410143/in/photostreamhttp://www.flickr.com/photos/youngkyi/8531410161/in/photostreamThank you very much!Tian    

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[-may]

Thank you Michael S. and Michael C.!  Your answers helped and I see that in addition to the brightness problem, there is also some movement of the features(fibrils), which weakened the disappearing effect and made the comparison more complicated.

Do you know a way to adjust two images with the same contrast or brightness?  

Thank you!

Tian

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[-may]

Also, does it make a difference whether the images are 8-bit, 16-bit or 32-bit?

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