Posted by
Dimiter Prodanov (imec) on
URL: http://imagej.273.s1.nabble.com/Is-manual-thresholding-methods-accepted-by-scientific-journals-tp5010814p5010839.html
Dear all,
This is an interesting question. For a starter, there are many different types of automatic thresholding each with their own assumptions.
There is no universal thresholding because the images statistics differ very much form the type of image (i.e. fluorescence, bright field, derivative) illumination or acquisition conditions and the properties of the background/scene.
So what you can do is try out several algos until you find the best for your data and then tweak it manually.
For most cases I have given up intensity thresholding years ago.
Nowadays I am mostly doing image segmentation using texture features or second order statistics.
Best regards,
Dimiter
-----Original Message-----
From: ImageJ Interest Group [mailto:
[hidden email]] On Behalf Of IMAGEJ automatic digest system
Sent: 07 December, 2014 6:00
To:
[hidden email]
Subject: IMAGEJ Digest - 5 Dec 2014 to 6 Dec 2014 (#2014-353)
There are 17 messages totaling 1178 lines in this issue.
Topics of the day:
1. imaging enhancements (2)
2. How to use setLocation and measure
3. Hough transform problem Edge detection
4. Where can I get Adaptive Histogram Equalization (AHE) Plugin in ImageJ
5. Is manual thresholding methods accepted by scientific journals? (7)
6. flip ROI selection (3)
7. Latest Bio-Formats in Fiji
8. Error when using BioFormats Import for Macro
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Date: Fri, 5 Dec 2014 22:33:24 -0800
From: Mohammad Faizal Hassan <
[hidden email]>
Subject: Re: imaging enhancements
Hi Phanikanth ,
For AHE and CLAHE, you can download them from this link respectively 1.
http://svg.dmi.unict.it/iplab/imagej/Plugins/Forensics/Histogram%20Equalization/HistogramEqualization.html2.
http://rsbweb.nih.gov/ij/plugins/clahe/index.htmlBy the way, I also have a problem in getting the AHE plugin that can be implemented in the ImageJ software. I have been searching it from many sources in the internet but still did not find any. It is much appreciated if you can tell me from where I can get the plugin of AHE for free in the internet.
Looking forward for your reply. Please help me. Thank you in advance.
-----
Mohammad Faizal Hassan
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Date: Sat, 6 Dec 2014 10:05:01 +0200
From: Avital Steinberg <
[hidden email]>
Subject: Re: How to use setLocation and measure
Thanks, but I don't want to create thousands of ROIs. I succeeded in moving the ROI around using:
rm.select(img,selectIndex);
rm.translate(offsetX,offsetY);
So the selected ROI has been moved to the place I wanted to move it to. The Image Plus object is associated with the ROI I'm moving around. Therefore,
using:
ImageStatistics stats = img.getStatistics();
Works well. But - I'd like to hide the image, and img is the Image Plus object. So I would have liked to work on the ImageProcessor object, like
this:
ImageProcessor ip = img.getProcessor();
ImageStatistics stats = ImageStatistics.getStatistics(ip, MEAN, cal);
But this gives me strange values.
Any idea how to link the Image Processor object with the currently selected ROI? The ImagePlus object is linked to the selected ROI.
Thank you,
Avital
On Fri, Dec 5, 2014 at 11:33 PM, Rasband, Wayne (NIH/NIMH) [E] <
[hidden email]> wrote:
> On Dec 5, 2014, at 12:24 PM, Avital Steinberg
> <
[hidden email]>
> wrote:
> >
> > Hi,
> > I am trying to move an ROI around in a java plugin and measure the
> > mean intensity. I am measuring the ImageStatistics on an
> > ImageProcessor,
> since I
> > don't want to display the image. The problem is that it is not
> > actually moving the ROI around. Here is my code: (img is ImagePlus)
>
> Create a new ROI for each location. The following example measures the
> mean of a circular ROI at 10 different locations.
>
> -wayne
>
> img = IJ.createImage("Untitled", "16-bit ramp", 500, 500, 1);
> w = h = 50;
> for (i=0; i<10; i++) {
> roi = new OvalRoi(i*50, i*50, w, h);
> img.setRoi(roi);
> stats = img.getStatistics();
> IJ.log(i+" "+stats.mean);
> }
>
>
> > I am giving a simple example with two different locations, but my
> > purpose is to move the ROI to many different locations and measure
> > the mean intensities.
> >
> > ImageProcessor ip = img.getProcessor(); Calibration cal =
> > img.getCalibration();
> > img.setRoi(new OvalRoi(598, 380, 32, 59)); Roi roi =
> > img.getRoi();
> > ImageStatistics stats = ImageStatistics.getStatistics(ip,
> > MEAN, cal); IJ.log("The mean intensity is: " + stats.mean);
> > roi.setLocation(1056, 304); stats =
> > ImageStatistics.getStatistics(ip, MEAN, cal); IJ.log("The mean
> > intensity is now: " + stats.mean);
> >
> > How should I change my code so that it would really move the ROI to
> > a new location and measure the mean intensity in this location?
> >
> > Thanks,
> > Avital
>
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Date: Sat, 6 Dec 2014 06:52:03 -0500
From: Nizar SGHAIER <
[hidden email]>
Subject: Re: Hough transform problem Edge detection
Good afternoon,
thank you all for your replies, so I am trying to detect edges and not circles, and I have used the Burger Hough Linear transform and I did not found the disired result, I am attaching some pictures to this e-mail to explain more what I am looking for. Big thanks To you Wilhelm for the plugin posted on your dropbox, I will test it today and tell you if it returns the desired results.
I am attaching images and the found java source code that gave me the disired results.
PS :
the firth link contains the java source code you can put the two files together in the same directory and compile and run using ImageJ to get it work.
The fifth link conatins the obtained results using the java source code.
thank you all
cheers
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Date: Sat, 6 Dec 2014 04:39:24 -0800
From: Mohammad Faizal Hassan <
[hidden email]>
Subject: Where can I get Adaptive Histogram Equalization (AHE) Plugin in ImageJ
Hi,
I am Faizal Hassan a final year student from Universiti Teknologi MARA, Malaysia. Currently I am doing my final year project about enhancement of medical images using *Adaptive Histogram Equalization (AHE) *in *ImageJ
software* . However, I have a difficulty in getting the AHE plugin that can be implemented in the software. I have been searching it from many sources in the internet but still did not find any. It is much appreciated if you can tell me from where I can get the plugin of AHE for free in the internet.
I really need your help.
FYI, I have found a plugin named Histogram Equalization (HE) that can be used in ImageJ (I have given the URL below) and I wish I could get the same thing for the AHE plugin. Looking forward for your reply. Please help me.
Thank you in advance.
*The HE URL: *
http://svg.dmi.unict.it/iplab/imagej/Plugins/Forensics/Histogram%20Equalization/HistogramEqualization.html-----
Mohammad Faizal Hassan
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Date: Sat, 6 Dec 2014 15:00:22 +0000
From: Anders Lunde <
[hidden email]>
Subject: Is manual thresholding methods accepted by scientific journals?
Dear mailing list,
I have developed a nice macro for identifying colocalized signals for z-stack confocal images with multiple channels/colors. However, my advisor/professor has now come to question my method for setting a threshold for signal/no-signal in the infividual channels.
My manual method has been to simply raise the threshold above what I relatively confidently can see is background, like large areas with no apparent staining. The reason I did it manually is because when I played around with the automatic thresholding methods in ImageJ I decided that they were not any better than manual and could be subject to mistakes.
My supervisor now feels that this sounds too subjective and would not look good in a paper. He therefore asked me to try to find a way that was more guided e.g. by the histogram or something, anything that is less subjective (not sure if he is worried about accuracy or how it sounds in a paper).
What is the current standard for this kind of analysis in scientific journals, in particular with regards to the acceptability of manual thresholding of immunofluorescent brain sections stained with various antibodies (and nuclear markers and neuron trancers)? Is there a preference for automated, manual or some hybrid methods? Could I "get-away" with something like this: "Thresholds were set manually at a level that excluded most pixels in assumed background areas. Inspection of the assigned threshold level in the ImageJ intensity histogram showed that the thresholds were set at where the main peak (background pixels) started to or had reached a minimum value."
Image set that Im working on:
I am working with images of brain sections with 4 colors/channels: nuclear stain, two immunofluorescence staining for transciption factors (nuclear localization), and a retrograde nerve cell staning (nuclear + cytoplasm staining).
Greateful for any advice!
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Date: Sat, 6 Dec 2014 18:24:32 +0300
From: Василий Попков <
[hidden email]>
Subject: Re: Is manual thresholding methods accepted by scientific journals?
Very interesting question indeed. From my experience I know, that journals lika PNAS can get along with manual thresholding. At least if it is not the very core of a work.
On the other hand I myself always shift between auto and manual method, trying to find balance between subjectivity and well... more accurate threshold.
One very serious professor in this field have given me advice to threshold each image with the same numbers for each experiment, obtained from image of "empty view". Maybe this advice can be applied to your work too.
2014-12-06 18:00 GMT+03:00 Anders Lunde <
[hidden email]>:
> Dear mailing list,
>
> I have developed a nice macro for identifying colocalized signals for
> z-stack confocal images with multiple channels/colors. However, my
> advisor/professor has now come to question my method for setting a
> threshold for signal/no-signal in the infividual channels.
>
> My manual method has been to simply raise the threshold above what I
> relatively confidently can see is background, like large areas with no
> apparent staining. The reason I did it manually is because when I
> played around with the automatic thresholding methods in ImageJ I
> decided that they were not any better than manual and could be subject to mistakes.
>
> My supervisor now feels that this sounds too subjective and would not
> look good in a paper. He therefore asked me to try to find a way that
> was more guided e.g. by the histogram or something, anything that is
> less subjective (not sure if he is worried about accuracy or how it sounds in a paper).
>
> What is the current standard for this kind of analysis in scientific
> journals, in particular with regards to the acceptability of manual
> thresholding of immunofluorescent brain sections stained with various
> antibodies (and nuclear markers and neuron trancers)? Is there a
> preference for automated, manual or some hybrid methods? Could I
> "get-away" with something like this: "Thresholds were set manually at
> a level that excluded most pixels in assumed background areas.
> Inspection of the assigned threshold level in the ImageJ intensity
> histogram showed that the thresholds were set at where the main peak
> (background pixels) started to or had reached a minimum value."
>
> Image set that Im working on:
>
> I am working with images of brain sections with 4 colors/channels:
> nuclear stain, two immunofluorescence staining for transciption
> factors (nuclear localization), and a retrograde nerve cell staning
> (nuclear + cytoplasm staining).
>
> Greateful for any advice!
>
> --
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http://imagej.nih.gov/ij/list.html>
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Date: Sat, 6 Dec 2014 10:27:01 -0500
From: Liz Bless <
[hidden email]>
Subject: flip ROI selection
Hello - I have images of brain sections - sometimes the right hemisphere and sometimes the left hemisphere. I have an ROI that I would like to analyze. How can I get my ROI from the left hemisphere to flip (not rotate) so that it fits onto the right hemisphere? In other words I would like a mirror image of the ROI I have made. Any advice would be very helpful!
Thank you
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Date: Sat, 6 Dec 2014 19:50:58 +0300
From: Василий Попков <
[hidden email]>
Subject: Re: flip ROI selection
Maybe it would be easier to mirior image itself?
2014-12-06 18:27 GMT+03:00 Liz Bless <
[hidden email]>:
> Hello - I have images of brain sections - sometimes the right
> hemisphere and sometimes the left hemisphere. I have an ROI that I
> would like to analyze. How can I get my ROI from the left hemisphere
> to flip (not rotate) so that it fits onto the right hemisphere? In
> other words I would like a mirror image of the ROI I have made. Any advice would be very helpful!
> Thank you
>
> --
> ImageJ mailing list:
http://imagej.nih.gov/ij/list.html>
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Date: Sat, 6 Dec 2014 12:08:51 -0500
From: Tiago Ferreira <
[hidden email]>
Subject: Re: flip ROI selection
Dear Liz,
On Dec 6, 2014, at 10:27, Liz Bless <
[hidden email]> wrote:
> I would like a mirror image of the ROI I have made.
The two macros below would do it:
Install them, create a selection, and press F1/F2 to obtain dorsal-caudal/ventral-dorsal mirrors of the active ROI.
These used to be present in earlier versions of the ROI Manager Tools[1], but somehow got removed as were deemed too specific (they were mainly used for manual quantification of in-situ slices). They could be re-introduced if you think they are of broader scope. In that case, they would be made available through the BAR update site[2].
To install: Copy the snippet below, paste it into ImageJ, then used the Install command in the editor window.
Hope it helps,
-tiago
[1]
http://imagej.net//plugins/roi-manager-tools/[2]
http://fiji.sc/BAR// Begin of snippet
macro "Contralateral in X [F1]" {
mirrorROI(-1, 1);
}
macro "Contralateral in Y [F2]" {
mirrorROI(1, -1);
}
function mirrorROI(fx, fy) {
roi = selectionType();
if (roi>8 || roi<0)
exit("Cannot mirror current selection");
getSelectionBounds(x, y, width, height);
originalX = x;
originalY = y;
getSelectionCoordinates(x, y);
aX= newArray(x.length);
aY= newArray(x.length);
for (i=0; i<x.length; i++) {
aX[i] = fx*x[i];
aY[i] = fy*y[i];
}
makeSelection(roi, aX, aY);
setSelectionLocation(originalX, originalY); } // End of snippet
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Date: Sat, 6 Dec 2014 13:26:07 -0500
From: "MrScienctistMan (Original poster)" <
[hidden email]>
Subject: Re: Is manual thresholding methods accepted by scientific journals?
I guess for example the triangle method in ImageJ looks pretty okay. Would it be more aceptable to use for example this instead of manual threshold?
Im not quite sure what you mean about empty image. Do you mean a non-stained negative control?
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Date: Sat, 6 Dec 2014 19:27:34 +0200
From: Aryeh Weiss <
[hidden email]>
Subject: Re: Latest Bio-Formats in Fiji
On 12/6/14, 12:16 AM, Curtis Rueden wrote:
> Hi Aryeh,
>
> > About two weeks ago, when 5.0.6 was announced, I allowed the Fiji
> > updater to update formats-gpl. The updated version correctly read my
> > multifield ND2, so I thought that this bug was resolved. However,
> > yesterday, following an update, the problem reappeared. Again,the
> > workaround was to use the 5.0.2 version of formats-gpl.
>
> That is very odd. It would be great if you could somehow isolate the
> difference between the working version of Fiji vs. the non-working
> one—especially if they both used Bio-Formats 5.0.6.
>
I checked my backups, and I have been unable to find a working updated bioformats-gpl in my recent backups. This most likely means that I was mistaken about the problem being temporarily fixed.
> What happens if you enable the Bio-Formats update site? Still non-working?
>
It fails both ways.
Here is the info form teh advanced mode of the updater for the two
bioformats-gpl files:
bioformats from fiji site
bioformats-gpl-5.0.6
Modified 11 Nov 2014
bioformats from bioformats site
bioformats-gpl
Modified 04 Nov 2014
Both fail the same way.
> If you get a chance, you could also test with the "showinf" command
> line tool [1], which would rule out any Fiji-specific installation
> issues. And of course testing from multiple different machines might
> also shed some light on things.
>
The showinf output has the same error -- it is not Fiji specific.
I can also tell you that the same problem occurs on a Windows 7-64bit
OS, and the workaroudn is identical (fall back to gpl-5.0.2
As long as the workaround works, it is ok. However, that seems like
living on borrowed time...
> Regards,
> Curtis
>
Thank you for your attention to this.
Best regards,
--aryeh
> [1]
>
http://openmicroscopy.org/site/support/bio-formats5/users/comlinetools/>
> On Thu, Nov 27, 2014 at 1:45 AM, Aryeh Weiss <
[hidden email]
> <mailto:
[hidden email]>> wrote:
>
> On 10/6/14, 11:17 PM, Aryeh Weiss wrote:
>
> Hi Melissa
>
> On 10/6/14, 4:27 PM, Melissa Linkert wrote:
>
> Hi Aryeh,
>
> You are correct. We will be switching over to
> 5.1.x, but in the mean time
> it looks like the nd2 fix
> <
https://github.com/openmicroscopy/bioformats/commit/> 3c857c2e5743e9b23801408bd61de49697716b4d>
> you're looking for only affected the
> formats-gpl component. You should be
> able to just download that specific jar, and
> replace the
> formats-gpl-5.0.5.jar in your
> Fiji.app/jars/bioformats directory (the old
> version will just be called "formats-gpl.jar"
> if you still have the
> Bio-Formats update site enabled).
>
> How can I turn this into the formats-gpl.jar file?
> If I can do that, I will replace my version 5.0.2
> file, and see if it
> works properly
>
> Instead of compiling from source, you can also download
> the latest 5.1
> builds from:
>
>
http://ci.openmicroscopy.org/view/Bio-Formats/job/BIOFORMATS-5.1-latest/lastSuccessfulBuild/artifact/artifacts/>
>
> For anyone trying this though, please do note that mixing
> 5.1 and 5.0
> files is not supported and very unlikely to work, so you
> will need to
> update all files to 5.1. Be aware that this can cause
> problems with
> other plugins that use Bio-Formats, as there are API
> differences between
> the two versions (though we have not yet released 5.1.0).
>
>
> Thank you (and also Mark) for your replies.
> I downloaded the 5.1 builds for all of the jar files in the
> jars/bio-formats directory of my Fiji distribution.
> When I dragged one of my nd2 files into Fiji, bio-formats
> opened, but had the same bug as reported previously.
> When I restored the old bio-formats directory with the 5.0.2
> version of gpl, it worked properly.
> I did not try the 5.0.2 gpl with the 5.1 release, because of
> Melissa warning concerning mixing the two versions.
>
>
> Best regards
> --aryeh
>
>
> We are also considering this fix for a 5.0.6 release of
> Bio-Formats, which
> would eliminate the need to manually update files.
>
> Regards,
> -Melissa
>
>
> About two weeks ago, when 5.0.6 was announced, I allowed the Fiji
> updater to update formats-gpl.
> The updated version correctly read my multifield ND2, so I thought
> that this bug was resolved.
> However, yesterday, following an update, the problem reappeared.
> Again,the workaround was to use the 5.0.2 version of
> formats-gpl.
>
> I have not had a chance to search my recent backups for this file
> to see if I can determine exactly when it broke again -- I will
> try to do that.
>
>
> Best regards,
> --aryeh
>
>
>
> On Mon, Oct 06, 2014 at 07:52:39AM -0500, Mark Hiner wrote:
>
> Hi Aryeh,
>
> How can I turn this into the formats-gpl.jar file?
>
> Great question! I'm cc'ing the list because I think
> this information is of
> general interest.
>
> The Bio-Formats site has a page with instructions for
> downloading and
> building the jars from source:
>
https://www.openmicroscopy.org/site/support/bio-formats5/developers/building-bioformats.html>
>
> After cloning the Bio-Formats repository
> <
http://git-scm.com/book/en/Git-Basics-Getting-a-Git-Repository#Cloning-an-Existing-Repository>,
>
> you can either:
> * Check out a specific tag
> <
http://stackoverflow.com/questions/791959/download-a-specific-tag-with-git>
>
> or
> * Check out a specific commit
> <
http://stackoverflow.com/questions/2007662/rollback-to-an-old-commit-using-git>
>
>
> Or it should be fine to just build the "develop"
> branch, since the patch in
> question was merged.
>
> After building Bio-Formats, all the output .jars will
> be in the /artifacts
> sub-directory of your Bio-Formats checkout.
>
> Let me know if you have any more questions, and thank
> you for taking the
> time to test this!
>
> Regards,
> Mark
>
>
> On Sat, Oct 4, 2014 at 3:03 PM, Aryeh Weiss
> <
[hidden email] <mailto:
[hidden email]>> wrote:
>
> Hi Mark
>
> On 10/1/14, 10:21 PM, Mark Hiner wrote:
>
> Hi Christophe,
>
> As far as I understand, the current dailies
> are 5.0, not 5.1 yet
> You are correct. We will be switching over to
> 5.1.x, but in the mean time
> it looks like the nd2 fix
> <
https://github.com/openmicroscopy/bioformats/commit/> 3c857c2e5743e9b23801408bd61de49697716b4d>
> you're looking for only affected the
> formats-gpl component. You should be
> able to just download that specific jar, and
> replace the
> formats-gpl-5.0.5.jar in your
> Fiji.app/jars/bioformats directory (the old
> version will just be called "formats-gpl.jar"
> if you still have the
> Bio-Formats update site enabled).
>
> How can I turn this into the formats-gpl.jar file?
> If I can do that, I will replace my version 5.0.2
> file, and see if it
> works properly
>
> thanks and best regards
> --aryeh
>
>
>
>
>
>
> --
> Aryeh Weiss
> Faculty of Engineering
> Bar Ilan University
> Ramat Gan 52900 Israel
>
> Ph: 972-3-5317638
> FAX: 972-3-7384051
>
> --
> ImageJ mailing list:
http://imagej.nih.gov/ij/list.html>
>
--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel
Ph: 972-3-5317638
FAX: 972-3-7384051
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Date: Sat, 6 Dec 2014 21:53:11 +0300
From: Василий Попков <
[hidden email]>
Subject: Re: Is manual thresholding methods accepted by scientific journals?
I mean just to take an image of something what is "background" in general.
And than use this value to minus it from all image and count it like
thresholding. Depends on your object, I guess.
As for me, I have tested all thresholding methods and only Shanbhag works
quite well for me. Still, manual method was better :(
For now I am doing so: find manually threshold for 1 image and than use
exactly the same values for all images in experiment. My objects are
supposed to have the same noise and background levels in 1 experiment.
2014-12-06 21:26 GMT+03:00 MrScienctistMan (Original poster) <
[hidden email]>:
> I guess for example the triangle method in ImageJ looks pretty okay. Would
> it be more aceptable to use for example this instead of manual threshold?
>
> Im not quite sure what you mean about empty image. Do you mean a
> non-stained negative control?
>
> --
> ImageJ mailing list:
http://imagej.nih.gov/ij/list.html>
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Date: Sat, 6 Dec 2014 14:35:13 -0500
From: Michael Schell <
[hidden email]>
Subject: Re: Is manual thresholding methods accepted by scientific journals?
In my experience, it is worth the time to find the best auto-threshold for your data set, and then stick to it. Of course, which method is “the best” varies with the images being analyzed. You just have to test them, do a few trial analyses, and find one that produces reasonable numbers that reflect what you see qualitatively. I agree with your advisor. It is not worth the risk using manual eyeball methods and then having a reviewer raise a problem with bias, whether it be real or perceived.
If you are analyzing a Z-stack, be sure you move to a bright image in the middle of the stack before applying the threshold to all images in the stack. If you apply a threshold to the first image in the stack and it has almost no signal, the thresholding will not be optimal.
A recent example of how we applied Triangle successfully for colocalization analysis can be found in Traffic 15:1344 (2014). This method was robust and the numbers made sense with respect to the biology. For some of the data, which were acquired using a different method, Triangle did not work well, but Otsu did. Your mileage may vary.
Michael
> On Dec 6, 2014, at 10:00 AM, Anders Lunde <
[hidden email]> wrote:
>
> Dear mailing list,
>
> I have developed a nice macro for identifying colocalized signals for
> z-stack confocal images with multiple channels/colors. However, my
> advisor/professor has now come to question my method for setting a
> threshold for signal/no-signal in the infividual channels.
>
> My manual method has been to simply raise the threshold above what I
> relatively confidently can see is background, like large areas with no
> apparent staining. The reason I did it manually is because when I played
> around with the automatic thresholding methods in ImageJ I decided that
> they were not any better than manual and could be subject to mistakes.
>
> My supervisor now feels that this sounds too subjective and would not look
> good in a paper. He therefore asked me to try to find a way that was more
> guided e.g. by the histogram or something, anything that is less subjective
> (not sure if he is worried about accuracy or how it sounds in a paper).
>
> What is the current standard for this kind of analysis in scientific
> journals, in particular with regards to the acceptability of manual
> thresholding of immunofluorescent brain sections stained with various
> antibodies (and nuclear markers and neuron trancers)? Is there a preference
> for automated, manual or some hybrid methods? Could I "get-away" with
> something like this: "Thresholds were set manually at a level that
> excluded most pixels in assumed background areas. Inspection of the
> assigned threshold level in the ImageJ intensity histogram showed that the
> thresholds were set at where the main peak (background pixels) started to
> or had reached a minimum value."
>
> Image set that Im working on:
>
> I am working with images of brain sections with 4 colors/channels: nuclear
> stain, two immunofluorescence staining for transciption factors (nuclear
> localization), and a retrograde nerve cell staning (nuclear + cytoplasm
> staining).
>
> Greateful for any advice!
>
> --
> ImageJ mailing list:
http://imagej.nih.gov/ij/list.html--
ImageJ mailing list:
http://imagej.nih.gov/ij/list.html------------------------------
Date: Sat, 6 Dec 2014 20:17:46 +0000
From: Jeremy Adler <
[hidden email]>
Subject: Re: Is manual thresholding methods accepted by scientific journals?
Segmenting images with multiple features is a problem
Manual thresholding provides scope for bias -
But blinded manual thresholding, when the person setting the threshold is unaware of which experimental group the images belong, is acceptable. I would also suggest recording the thresholds and making these and the original images available after publication.
A related question is which images, of all those that could have been acquired, were used in the analysis and how were they chosen. This should form part of the methods section but is rarely described.
________________________________________
From: ImageJ Interest Group [
[hidden email]] on behalf of Anders Lunde [
[hidden email]]
Sent: 06 December 2014 16:00
To:
[hidden email]
Subject: Is manual thresholding methods accepted by scientific journals?
Dear mailing list,
I have developed a nice macro for identifying colocalized signals for
z-stack confocal images with multiple channels/colors. However, my
advisor/professor has now come to question my method for setting a
threshold for signal/no-signal in the infividual channels.
My manual method has been to simply raise the threshold above what I
relatively confidently can see is background, like large areas with no
apparent staining. The reason I did it manually is because when I played
around with the automatic thresholding methods in ImageJ I decided that
they were not any better than manual and could be subject to mistakes.
My supervisor now feels that this sounds too subjective and would not look
good in a paper. He therefore asked me to try to find a way that was more
guided e.g. by the histogram or something, anything that is less subjective
(not sure if he is worried about accuracy or how it sounds in a paper).
What is the current standard for this kind of analysis in scientific
journals, in particular with regards to the acceptability of manual
thresholding of immunofluorescent brain sections stained with various
antibodies (and nuclear markers and neuron trancers)? Is there a preference
for automated, manual or some hybrid methods? Could I "get-away" with
something like this: "Thresholds were set manually at a level that
excluded most pixels in assumed background areas. Inspection of the
assigned threshold level in the ImageJ intensity histogram showed that the
thresholds were set at where the main peak (background pixels) started to
or had reached a minimum value."
Image set that Im working on:
I am working with images of brain sections with 4 colors/channels: nuclear
stain, two immunofluorescence staining for transciption factors (nuclear
localization), and a retrograde nerve cell staning (nuclear + cytoplasm
staining).
Greateful for any advice!
--
ImageJ mailing list:
http://imagej.nih.gov/ij/list.html--
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Date: Sat, 6 Dec 2014 15:27:44 -0500
From: John Oreopoulos <
[hidden email]>
Subject: Re: Is manual thresholding methods accepted by scientific journals?
Dear Anders,
There are two very good publications on this topic that come to mind:
Rossner, M., and Yamada, K. M. (2004). What's in a picture? The temptation of image manipulation. J. Cell Biol., 166, 11-15.
Cromey, D. (2010). Avoiding twisted pixels: Ethical guidelines for the appropriate use and manipulation of scientific digital images.
I can't find any specific comments about thresholding, but the "5th commandment" listed in Cromey's publications states:
5. Digital Images that will be Compared to one Another Should be Acquired under Identical Conditions, and any Post-acquisition Image Processing Should also be Identical:
"When images are to be compared to one another, the processing of the individual images should be identical. This includes acquisition techniques such as background subtraction or white-level balancing, which should be documented in the methods section. The same principle applies to publication figures, especially if multiple images will be published together in a single figure. This assists the reader in understanding how each image relates to the others in the group. Individual images within a figure should only be processed differently if there are compelling reasons to do so. In such cases, the differences must be explained in the methods section or the figure legend. Honesty, and completeness, are the best policies."
I think it is implied here that having an automatic / computer-guided thresholding workflow as opposed to one that requires human intervention is preferred so that all data is treated equally. But, I think as long as you are clear in your methods section about why and how you chose manual thresholding and state that you have done so, I think this would be acceptable in a paper. Ask yourself, however, would someone else have thresholded your images the same way you did? If not, they might get different results and interpretation of your analysis. Bottom line, you should supply as much information describing how you performed your image analysis especially if the crux of the paper hinges on it. Providing raw data in the supplementary materials is another way for reviewers to check the validity of your manual thresholding argument.
John Oreopoulos
On 2014-12-06, at 10:00 AM, Anders Lunde wrote:
> Dear mailing list,
>
> I have developed a nice macro for identifying colocalized signals for
> z-stack confocal images with multiple channels/colors. However, my
> advisor/professor has now come to question my method for setting a
> threshold for signal/no-signal in the infividual channels.
>
> My manual method has been to simply raise the threshold above what I
> relatively confidently can see is background, like large areas with no
> apparent staining. The reason I did it manually is because when I played
> around with the automatic thresholding methods in ImageJ I decided that
> they were not any better than manual and could be subject to mistakes.
>
> My supervisor now feels that this sounds too subjective and would not look
> good in a paper. He therefore asked me to try to find a way that was more
> guided e.g. by the histogram or something, anything that is less subjective
> (not sure if he is worried about accuracy or how it sounds in a paper).
>
> What is the current standard for this kind of analysis in scientific
> journals, in particular with regards to the acceptability of manual
> thresholding of immunofluorescent brain sections stained with various
> antibodies (and nuclear markers and neuron trancers)? Is there a preference
> for automated, manual or some hybrid methods? Could I "get-away" with
> something like this: "Thresholds were set manually at a level that
> excluded most pixels in assumed background areas. Inspection of the
> assigned threshold level in the ImageJ intensity histogram showed that the
> thresholds were set at where the main peak (background pixels) started to
> or had reached a minimum value."
>
> Image set that Im working on:
>
> I am working with images of brain sections with 4 colors/channels: nuclear
> stain, two immunofluorescence staining for transciption factors (nuclear
> localization), and a retrograde nerve cell staning (nuclear + cytoplasm
> staining).
>
> Greateful for any advice!
>
> --
> ImageJ mailing list:
http://imagej.nih.gov/ij/list.html--
ImageJ mailing list:
http://imagej.nih.gov/ij/list.html------------------------------
Date: Sat, 6 Dec 2014 14:40:50 -0800
From: JNHBoon <
[hidden email]>
Subject: Error when using BioFormats Import for Macro
I'm trying to open a stack of images (400) in .dm4 format using the
BioFormats plugin. Tried using the macro recorder and this is what it gave
me:
run("Bio-Formats", "open=[D:\\oxygen_3e-5_1 (missing Minute
3)\\Hour_00\\Minute_00\\Second_00\\oxygen_3e-5_1_Hour_00_Minute_00_Second_00_Frame_0000.dm4]
color_mode=Default group_files open_all_series view=Hyperstack
stack_order=XYCZT use_virtual_stack axis_1_number_of_images=400
axis_1_axis_first_image=0 axis_1_axis_increment=1 file=[]
pattern=[D:\\\\oxygen_3e-5_1 (missing Minute
3)\\\\Hour_00\\\\Minute_00\\\\Second_00\\\\oxygen_3e-5_1_Hour_00_Minute_00_Second_00_Frame_0<000-399>.dm4]");
Unfortunately, when I tried running the macro, it gave me this error. Does
anyone know what might be wrong here?
java.lang.NullPointerException
at ij.Macro.trimKey(Macro.java:154)
at ij.gui.GenericDialog.getNextBoolean(GenericDialog.java:936)
at
loci.plugins.in.FilePatternDialog.harvestResults(FilePatternDialog.java:192)
at loci.plugins.in.ImporterDialog.showDialog(ImporterDialog.java:83)
at
loci.plugins.in.ImporterPrompter.promptFilePattern(ImporterPrompter.java:135)
at
loci.plugins.in.ImporterPrompter.statusUpdated(ImporterPrompter.java:84)
at
loci.plugins.in.ImportProcess.notifyListeners(ImportProcess.java:475)
at loci.plugins.in.ImportProcess.step(ImportProcess.java:751)
at loci.plugins.in.ImportProcess.execute(ImportProcess.java:146)
at loci.plugins.in.Importer.showDialogs(Importer.java:141)
at loci.plugins.in.Importer.run(Importer.java:79)
at loci.plugins.LociImporter.run(LociImporter.java:81)
at ij.IJ.runUserPlugIn(IJ.java:202)
at ij.IJ.runPlugIn(IJ.java:166)
at ij.Executer.runCommand(Executer.java:131)
at ij.Executer.run(Executer.java:61)
at ij.IJ.run(IJ.java:275)
at ij.macro.Functions.doRun(Functions.java:591)
at ij.macro.Functions.doFunction(Functions.java:89)
at ij.macro.Interpreter.doStatement(Interpreter.java:226)
at ij.macro.Interpreter.doStatements(Interpreter.java:214)
at ij.macro.Interpreter.run(Interpreter.java:111)
at ij.macro.Interpreter.run(Interpreter.java:81)
at ij.macro.Interpreter.run(Interpreter.java:92)
at ij.plugin.Macro_Runner.runMacro(Macro_Runner.java:153)
at ij.IJ.runMacro(IJ.java:119)
at ij.IJ.runMacro(IJ.java:108)
at net.imagej.legacy.IJ1Helper.runMacro(IJ1Helper.java:782)
at
net.imagej.legacy.plugin.IJ1MacroEngine.eval(IJ1MacroEngine.java:116)
at
net.imagej.legacy.plugin.IJ1MacroEngine.eval(IJ1MacroEngine.java:156)
at org.scijava.script.ScriptModule.run(ScriptModule.java:175)
at org.scijava.module.ModuleRunner.run(ModuleRunner.java:167)
at org.scijava.module.ModuleRunner.call(ModuleRunner.java:126)
at org.scijava.module.ModuleRunner.call(ModuleRunner.java:65)
at
org.scijava.thread.DefaultThreadService$2.call(DefaultThreadService.java:164)
at java.util.concurrent.FutureTask.run(FutureTask.java:262)
at
java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1145)
at
java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:615)
at java.lang.Thread.run(Thread.java:745)
--
View this message in context:
http://imagej.1557.x6.nabble.com/Error-when-using-BioFormats-Import-for-Macro-tp5010825.htmlSent from the ImageJ mailing list archive at Nabble.com.
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Date: Sat, 6 Dec 2014 22:19:45 -0500
From: Stephan Saalfeld <
[hidden email]>
Subject: Re: imaging enhancements
Hi Mohammed and Phanikanth,
CLAHE is AHE with a contrast limit. That is, if you set the contrast
limit in CLAHE to a very high value (the maximum intensity value of your
image is sufficient), you get AHE. That said, all you need is the CLAHE
implementation available in Fiji:
http://fiji.sc/Enhance_Local_Contrast_%28CLAHE%29Best,
Stephan
On Fri, 2014-12-05 at 22:33 -0800, Mohammad Faizal Hassan wrote:
--
Stephan Saalfeld, Ph.D.
Group Leader
Janelia Farm Research Campus
19700 Helix Drive | Ashburn, VA 20147
Phone: 571-209-4184 | Fax: 571-209-4946
[hidden email]
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End of IMAGEJ Digest - 5 Dec 2014 to 6 Dec 2014 (#2014-353)
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