brightness change correction

[Thorsten Mielke]

Dear all,

I am using Fiji/Trakem2 to stitch TEM images (mrc-format, 32 bit real,
map mode 2) automatically recorded from thin sections using Leginon.
Since the sequential data collection from several positions typically
takes several hours (up to > 10 hours ), we sometimes observe intensity
changes, most likely due an intensity change of the LaB6 cathode or a
change in beam diameter over time. This results in a gray level gradient
within the montages. We tried to use "Histogram matching" under "Image
-> Adjust ->  Bleach Correction" to normalize the intensity. However, it
seems that this works only for 16 bit mrc-stacks, which we obtain using
programs such as IMOD or Eman2.

Is there an easy way to convert 32 bit mrc to 16 bit mrc files within
Fiji? Is it possible to run the bleach correction (or any better tool?)
within individual TrakEM2 layers comprising up to ~1000 images?

Thanks for comments and help,

Thorsten

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[Cammer, Michael]

We correct our EM images with the Float Stack macro below.  Sometimes we tweak "saturated=0.35");"
Regards,
Michael

//---------------------------------------------------------
//Kenton Arkill [[hidden email]]
//Floating means that each image has the same mean pixel value and the same standard deviation.
//I find it really useful as it means the same threshold can often be used on all the images. Here is my macro,
//it floats the stack to the values of the first slice (so make sure it is a good image):

macro "Float the stack"{
run("32-bit");
setSlice(1);
getRawStatistics(nPixels, meanref, min, max, stdref, histogram);
for (i = 1; i <= nSlices; i++) {
       setSlice(i);
       getRawStatistics(nPixels, meani, min, max, stdi, histogram);
       run("Subtract...", "value="+meani+" slice");
       run("Divide...", "value="+stdi+" slice");
       run("Multiply...", "value="+stdref+" slice");
       run("Add...", "value="+stdref+" slice");
}
run("Enhance Contrast", "saturated=0.35");
setSlice(1);
}

_________________________________________
Michael Cammer, Optical Microscopy Specialist
http://ocs.med.nyu.edu/microscopy
Lab: [phone]  Cell: [phone]

________________________________________
From: ImageJ Interest Group [[hidden email]] on behalf of Thorsten Mielke [[hidden email]]
Sent: Wednesday, August 27, 2014 11:20 AM
To: [hidden email]
Subject: brightness change correction

Dear all,

I am using Fiji/Trakem2 to stitch TEM images (mrc-format, 32 bit real,
map mode 2) automatically recorded from thin sections using Leginon.
Since the sequential data collection from several positions typically
takes several hours (up to > 10 hours ), we sometimes observe intensity
changes, most likely due an intensity change of the LaB6 cathode or a
change in beam diameter over time. This results in a gray level gradient
within the montages. We tried to use "Histogram matching" under "Image
-> Adjust ->  Bleach Correction" to normalize the intensity. However, it
seems that this works only for 16 bit mrc-stacks, which we obtain using
programs such as IMOD or Eman2.

Is there an easy way to convert 32 bit mrc to 16 bit mrc files within
Fiji? Is it possible to run the bleach correction (or any better tool?)
within individual TrakEM2 layers comprising up to ~1000 images?

Thanks for comments and help,

Thorsten

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