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Dear Glen,

Indeed, I was using an 8 bit images, the sample Confocal-Series.tif. But I was not aware of this difference in thresholding between 8 bit and 16 bit images...

Cheers

Kees

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Glen MacDonald
Sent: 22 March 2012 18:25
To: [log in to unmask]
Subject: Re: 'smart' average z-projection

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Dear Kees,
did you run this on 8-bit images or 16-bit? 'run("Make Binary", "black");' and 'run("Convert to Mask", "  black");' behave similarly on 16-bit images, but run("Make Binary", "black"); on an 8-bit does not set to 0+255. However,  dividing the Make Binary 8-bit  image by 255 effectively sets the image to 0+1 due to rounding.  My macro was written for 16-bit images, but I'll change it to prevent future issues if used with 8-bit.  thanks for pointing this out.  

Regards,
Glen
On Mar 22, 2012, at 4:28 AM, Straatman, Kees R. (Dr.) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> Dear Philipp, Glen and list,
> 
> I tried the code Glen gave but the macro did not behave as I expected, at least not in my hands. Problem was the line 'run("Make Binary", "black");'  Changes in threshold settings were not reflected in the final image. I made a small change what worked for me. Just in case others have the same problem:
> 
> macro Smart_Average{
> 	stackid=getImageID;
> 	no=nSlices;
> 	stackname=getTitle;
> 	run("Duplicate...", "title=[dup] duplicate range=1-no");
> 	setSlice((floor(no/2)));
> 	run("Threshold...");
> 	waitForUser("Set Threshold then click 'OK'.");
> 	setBatchMode(true);
> 	run("Convert to Mask", "  black");
> 	run("Divide...", "value=255 stack");
> 	rename("divided");
> 	bindupid=getImageID;
> 	imageCalculator("Multiply create stack", stackid ,bindupid);
> 	run("Z Project...", "start=1 stop=no projection=[Sum Slices]");
> 	rename(stackname+"sum");
> 	sumid=getImageID;
> 	setBatchMode(false);
> 	selectImage(bindupid);//closing and renaming actions must occur outside of the batch mode
> 	close();
> }
> 
> Best wishes
> 
> Kees
> 
> 
> Dr Ir K.R. Straatman
> Senior Experimental Officer
> Centre for Core Biotechnology Services
> College of Medicine, Biological Sciences and Psychology
> University of Leicester
> 
> http://www.le.ac.uk/biochem/microscopy/home.html
> 
> 
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Philipp Bethge
> Sent: 21 March 2012 19:55
> To: [log in to unmask]
> Subject: Re: 'smart' average z-projection
> 
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> Dear Users,
> 
> Wow, thanks alot to everybody for the quick reply and the solutions offered.
> I tried out the one from Glen and it works very nice!
> 
> Thanks alot,
> 
> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> Dear Philipp,
>> Duplicate the stack, threshold for the structure then make the tholded stack binary
>> dividing by 255 converts the binarized stack from 0+255 to 0+1
>> multiply the 2 stacks and project the result.
>> this function does all this, choosing middle slice for setting the threshold
>> 
>> Maybe this is helpful,
>> 
>> function thresholdprojection(){
>> 	stackid=getImageID;
>> 	no=nSlices;
>> 	stackname=getTitle;
>> 	run("Duplicate...", "title=[dup] duplicate range=1-no");
>> 	setSlice((floor(no/2)));
>> 	run("Threshold...");
>> 	waitForUser("Set Threshold then click 'OK'.");
>> 	setBatchMode(true);
>> 	getThreshold(lower,upper);
>> 	run("Make Binary", "black");
>> 	run("Divide...", "value=255 stack");
>> 	rename("divided");
>> 	bindupid=getImageID;
>> 	imageCalculator("Multiply create stack", stackid ,bindupid);
>> 	run("Z Project...", "start=1 stop=no projection=[Sum Slices]");
>> 	rename(stackname+"sum");
>> 	sumid=getImageID;
>> 	setBatchMode(false);
>> 	selectImage(bindupid);//closing and renaming actions must occur outside of the batch mode
>> 	close();
>> }
>> 
>> Regards,
>> Glen
>> Glen MacDonald
>> Core for Communication Research
>> Virginia Merrill Bloedel Hearing Research Center
>> Box 357923
>> University of Washington
>> Seattle, WA 98195-7923  USA
>> (206) 616-4156
>> [log in to unmask]
>> 
>> 
>> 
>> 
>> 
>> 
>> 
>> 
>> On Mar 21, 2012, at 8:20 AM, Philipp Bethge wrote:
>> 
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>> 
>>> Dear all,
>>> 
>>> I was wondering if somebody has worked with a 'smart' average z-projection, that is a average projection of a z-stack that does not average of the entire stack size but only over the slides where a signal is actually present. So, for example, one has an oblique structure in a large stack, lets say the structure has a z-extension of 4 slides but the whole stack is 30 stacks large. As I understand the regular average z-projection algorithm in ImageJ, every pixel is summed and devided by the total number of slides in the stack. Then the signal of only 4 slides would be averaged over 30 slides, therefore reducing signal intensity dramatically without further reducing background noise.
>>> Of course, one would have to define 'signal' somehow, but I guess that could be done manually by binarization of the image slides individually.
>>> 
>>> Any suggestions welcome,
>>> 
>>> Thanks alot,
>>> 
>>> -- 
>>> Philipp Bethge
> 
> 
> -- 
> Philipp Bethge