Comet assay

[Hélio Miot]

Hi all,
I'm having some difficulties to
estimate the tail length from comet assay.
And can't segmentate the nucleus alone.

Is there any plugin?

Thanks,

Hélio A. Miot
Professor Assistente Doutor
Departamento de Dermatologia, S/N
Faculdade de Medicina da UNESP
Campus Universitário de Rubião Jr.
18618-000   -   Botucatu - SP
*******Nao imprima esse e-mail, economize papel, poupe árvores*******
----- Original Message -----
From: "IMAGEJ automatic digest system" <[hidden email]>
To: <[hidden email]>
Sent: Tuesday, July 10, 2007 1:00 AM
Subject: IMAGEJ Digest - 8 Jul 2007 to 9 Jul 2007 (#2007-178)

> There are 8 messages totalling 444 lines in this issue.
>
> Topics of the day:
>
>  1. Segmentation of granular "stuff"
>  2. Information from Macro (3)
>  3. stack threshold
>  4. ImageJ interaction: ROI Manager etc. (2)
>  5. ImageJ undo notification
>
> ----------------------------------------------------------------------
>
> Date:    Mon, 9 Jul 2007 14:29:15 +0200
> From:    Michael Schmid <[hidden email]>
> Subject: Re: Segmentation of granular "stuff"
>
> Hi Jolien,
>
> first concerning the uploaded images - the blanks in the filenames
> should be written "%20" in URLs. So, e.g., it is
>
> www.waisman.wisc.edu/cmn/mutant%20high%20load.tif
>
> Concerning faint images: There are plugins that may solve the
> problem, e.g. the Stack NOrmalizer
>   http://rsb.info.nih.gov/ij/plugins/normalizer.html
> and the Background Subtraction and Image Normalization plugin
>   http://rsb.info.nih.gov/ij/plugins/normalize.html
>
> Another way to do it might be saturating a small percentage
> of the pixels (Brightness&Contrast/Auto) and applying this as
> new grayscale (8-bit images only). The Macro Recorder will show
> you how this translates into macro code.
>
> If some images don't have the bright granular "stuff" at all,
> you may have to analyze at the histogram, e.g. determine the
> "mode" (grayvalue of the highest peak, usually some kind of
> background) and draw your conclusions from that (maybe divide
> by some value proportional to the "mode")
>
> Michael
> ________________________________________________________________
>
> On 6 Jul 2007, at 20:42, Jolien Connor wrote:
>
>> Hi,
>>
>> I am trying to do something similar to what many try
>> to do with thresholding -- find a way for the software
>> to pick out my pixels of interest away from the
>> background.  In my case, it is granular "stuff" that
>> are intracellular inclusions in the brain.  We have
>> many image montages taken of mouse brain sections and
>> we are trying to quantitate the percentage of specific
>> brain regions occupied by the inclusions (percent
>> volume).  Some of the monochrome images are here:
>>
>> www.waisman.wisc.edu/cmn/mutant high load.tif
>> www.waisman.wisc.edu/cmn/mutant high load and
>> faint.tif
>> www.waisman.wisc.edu/cmn/mutant low load and faint.tif
>> www.waisman.wisc.edu/cmn/control.tif
>>
>> They are very large since they are montages; contact
>> me at [hidden email] if you want them and
>> can't get them.
>>
>> If you look at the pictures of the mutants compared
>> with the control, you can see what they are -- the
>> bright granular stuff all over the place.  Cells that
>> contain the inclusions sometimes have processes that
>> show up, and we only want to pick out the brightest,
>> most granular stuff, which are the inclusions.  We
>> want a method that can tell the difference between
>> mutants and controls, as well as between high load
>> mutants and low load mutants (lots of them versus just
>> a few).
>>
>> The biggest problem right now seems to be that some of
>> our pictures are faint, possibly because the stain
>> weakened, our mercury bulb Atto Arc controller is
>> dying, or some other reason.  As humans, we can still
>> pick them out, but we don't have a thresholding
>> algorithm that can handle this problems.
>>
>> We have been using the Subtract Background function,
>> then thresholding the image, and can't get consistent
>> results.  We use a brightfield image that lines up
>> with each monochrome image to trace our regions, then
>> have ImageJ calculate the % Area occupied by pixels
>> above threshold.  There's no threshold we could pick
>> that would correctly differentiate between controls,
>> low load mutants and high load mutants.  Any ideas?
>>
>> Thanks,
>>
>> Jolien Connor
>> University of Wisconsin
>
> ------------------------------
>
> Date:    Mon, 9 Jul 2007 14:32:27 +0200
> From:    "Holmes, Charlotte" <[hidden email]>
> Subject: Information from Macro
>
> Hi
>
> I was wondering if there was an easy way for a plugin to get information
> from a macro?
>
> I have an old macro I use to put a grid on an image. I have now
> developed a plugin to help analyse the image with the grid on it however
> to go forward I really need the locations of the grid points.
> I could to this within the plugin but it would be a lot of work when I
> have the macro there with the information in it.
>
> Thanks
> Lottie
>
> ------------------------------
>
> Date:    Mon, 9 Jul 2007 14:55:01 +0200
> From:    Michael Schmid <[hidden email]>
> Subject: Re: Information from Macro
>
> Hi Charlotte,
>
> if the macro calls the plugin, you could simply use the
> GenericDialog in the plugin, and pass the values as parameters
> (run the plugin manually and use Macro>Record to see how to do
> this).
>
> In case your macro does not call the plugin, you can save the
> data as global variables (use the "var" macro statement) and
> make a wrapper macro for the plugin that calls the plugin with
> these variables passed to the GenericDialog.
>
> Otherwise, you have to store the values somewhere else, e.g.
> with macro function setMetadata into the ImagePlus, and use
>   String metadata = (String)imp.getProperty("Info");
> for non-stack images or
>   String  metadata = imp.getStack().getSliceLabel(imp.getCurrentSlice
> ());
> for stacks.
> Of course, you can also write the data into a file with a
> fixed name and read the file in the plugin.
> A disadvantage of these ways is that you will have to decode
> strings to get numbers in your plugin.
>
> Michael
> ________________________________________________________________
>
> On 9 Jul 2007, at 14:32, Holmes, Charlotte wrote:
>
>> Hi
>>
>> I was wondering if there was an easy way for a plugin to get
>> information
>> from a macro?
>>
>> I have an old macro I use to put a grid on an image. I have now
>> developed a plugin to help analyse the image with the grid on it
>> however
>> to go forward I really need the locations of the grid points.
>> I could to this within the plugin but it would be a lot of work when I
>> have the macro there with the information in it.
>>
>> Thanks
>> Lottie
>
> ------------------------------
>
> Date:    Mon, 9 Jul 2007 08:55:26 -0400
> From:    Ben Tupper <[hidden email]>
> Subject: Re: Information from Macro
>
> On Jul 9, 2007, at 08:32 AM, Holmes, Charlotte wrote:
>
>> Hi
>>
>> I was wondering if there was an easy way for a plugin to get
>> information
>> from a macro?
>>
>> I have an old macro I use to put a grid on an image. I have now
>> developed a plugin to help analyse the image with the grid on it
>> however
>> to go forward I really need the locations of the grid points.
>> I could to this within the plugin but it would be a lot of work when I
>> have the macro there with the information in it.
>
>
> Hello,
>
> You might want to look at IJ.runMacro() and IJ.runMacroFile(). Each
> of these will run the macro you specify, deliver arguments (as
> String) to the macro, and return a String value from the macro to the
> calling plugin.  I use this a lot with arguments (and return values)
> concatenated into a single string delimited by the "\n" newline
> character.  I often use the first line of the argument for processing
> flags - like if I want the macro to run in batch mode, etc.  If I
> need a number of processing flags in the first line, I concatenate
> those with tabs or commas.
>
> Here is a snippet from a macro that is called by a plugin....
>
>> arg = split(getArgument(),"\n");//split all the records, the first
>> of which holds processing info
>> pi = 3.141593;
>> // get the first line of flags etc.  The first line has booleans,
>> filepaths,
>> //  numbers etc. in tab-delimited fields
>>     s = split(arg[0], "\t");
>>     doVerbose = startsWith(s[0], "true");
>>     doBatchMode = startsWith(s[1], "true");
>>     doEnhance = startsWith(s[2], "true");
>>     doScalebar = startsWith(s[3], "true");
>>     cal = parseFloat(s[4]);
>>     name = s[5];
>>     dir = s[6];
>>     maskDir = s[7];
>>     doMask = !(startsWith(maskDir, "noMask"));
>>     outlineDir = s[8];
>>     doOutline = !(startsWith(outlineDir,"noOutline"));
>>     distNN = s[9];
>>     thresh = s[10];
>>     closeHoles = s[11];
>>     lightOrDark = s[12];
>>     minSize = pow(s[13]/2.,2) * pi;  //convert minESD to minArea
>>     maxSize= pow(s[14]/2., 2) * pi; //convert maxESD to maxArea
>>     slice = parseInt(s[s.length-1]);
>>
>> n = arg.length-1;// the number of records to process
>
>
> You probably get the idea. Hope that is what you are looking for.
>
> Cheers,
> Ben
>
> ------------------------------
>
> Date:    Mon, 9 Jul 2007 15:09:05 +0200
> From:    Michael Schmid <[hidden email]>
> Subject: Re: stack threshold
>
> Hi Cosmin,
>
> what about converting the threshold of the first slice into a
> selection
>   run("Create Selection");
> and then either replacing these pixels (or the non-selected ones,
> using "Make Inverse", depending on your needs) by some value
> that does not interfere with the analysis?
> E.g. "Clear", "Fill", run("Min...", "value=255 stack"); etc.
>
> Even without modifying the image, the selection will restrict
> the area for future measurements of other slices, so just creating
> the selection might be enough (e.g. if you use the histogram or
> getStatistics for analysis of the stack slices).
>
> Michael
> ________________________________________________________________
>
> On 6 Jul 2007, at 21:55, Cosmin Mihai wrote:
>
>> Hi,
>>
>> I have a question regarding thresholding of ImageJ stacks.
>> When using the threshold comand on the first slice, the rest of the
>> stack
>> is thresholded in the same limits. I am studying bleaching
>> processes and I
>> am interested in eliminating from my analysis pixels above a certain
>> value. The problem in the standard threshold is that the pixels
>> eliminated
>> from the first slice might be reintroduced in the later slices when
>> their
>> intensity falls in the threshold interval.
>> Is there any way to permanently eliminate those pixels from the whole
>> stack ?
>>
>> Thank you,
>> Cosmin
>
> ------------------------------
>
> Date:    Mon, 9 Jul 2007 16:19:32 +0200
> From:    Michael Schmid <[hidden email]>
> Subject: Re: ImageJ interaction: ROI Manager etc.
>
> On 5 Jul 2007, at 09:41, Karsten Rodenacker wrote:
>
>> Using the ROI Manger with display, the numbers in the display
>> usually don't correspond with the numbers of the ROI's in the list,
>> at least after having deleted any object from the list. Is that
>> intended?
> Hmm, the rois are not numbered in the roi manager, but have names
> that relate to their y and x coordinates. The labels are the position
> in the list of rois and updated correctly, as far as I see (ImageJ
> 1.38v; maybe you have an earlier version?)
>
>> Using the Wand on a mask image clicking into the background will
>> select the next most connected object on the right. Is that intended?
> It has been like this since the early days of ImageJ and I think
> everyone is used to it by now, even though it is different from the
> Wand of other programs.
>
>> Having a list of objects in the ROI manager, I would like to
>> correct one selected, either cut it into two or change it and than
>> update it in the ROI manager. My quite complicated way was:
>> 1. Select the object
>> 2. Change the mask image by drawing, deleting, etc.
>> 3. Remove the selection
>> 4. Multiple object select by doWand with shift if there are more
>> than one object after correction
>> 5. Add the object(s) to ROI Manager
>> 6. Find the formerly selected object and delete it in the ROI list
>> Another again complicated method is:
>> 1. correct the mask image
>> 2. (re)run analyze particles...
>> 3. control the objects
>> 4. if necessary go to 1.
>> Is there any idea to improve this proceeding?
> What about this:
>  1. select all items in ROI manager
>  2. ROI Manager More>>Combine (may be slow with many particles)
>  3. Create Mask
>  4. correct whatever you like (draw, delete, fill)
>  5. run "Analyze Particles" on the mask
>  6. delete everything in the ROI Manager
>  7. ROI Manager More>>Add Particles
>
> 1.-3. is not needed in case you have the mask of all objects already.
> ---
> Michael
>
> ------------------------------
>
> Date:    Mon, 9 Jul 2007 17:52:58 +0200
> From:    Karsten Rodenacker <[hidden email]>
> Subject: Re: ImageJ interaction: ROI Manager etc.
>
> Thank you Michael for explanations. Consider my questions as from a
> newbie.
>
> Am 09.07.2007 um 16:19 schrieb Michael Schmid:
>
>> On 5 Jul 2007, at 09:41, Karsten Rodenacker wrote:
>>
>>> Using the ROI Manger with display, the numbers in the display
>>> usually don't correspond with the numbers of the ROI's in the
>>> list, at least after having deleted any object from the list. Is
>>> that intended?
>> Hmm, the rois are not numbered in the roi manager, but have names
>> that relate to their y and x coordinates. The labels are the
>> position in the list of rois and updated correctly, as far as I see
>> (ImageJ 1.38v; maybe you have an earlier version?)
>>
> I mistook the first four character in the ROI list as number. I have
> the current ImageJ version, that live updater is very helpful.
> However, dealing with several hundred particles, it is cumbersome to
> relate the displayed number with the respective ROI.
>
>>> Using the Wand on a mask image clicking into the background will
>>> select the next most connected object on the right. Is that intended?
>> It has been like this since the early days of ImageJ and I think
>> everyone is used to it by now, even though it is different from the
>> Wand of other programs.
>>
> Ok, I did not trust my machine!
>
>>> Having a list of objects in the ROI manager, I would like to
>>> correct one selected, either cut it into two or change it and than
>>> update it in the ROI manager. My quite complicated way was:
>>> 1. Select the object
>>> 2. Change the mask image by drawing, deleting, etc.
>>> 3. Remove the selection
>>> 4. Multiple object select by doWand with shift if there are more
>>> than one object after correction
>>> 5. Add the object(s) to ROI Manager
>>> 6. Find the formerly selected object and delete it in the ROI list
>>> Another again complicated method is:
>>> 1. correct the mask image
>>> 2. (re)run analyze particles...
>>> 3. control the objects
>>> 4. if necessary go to 1.
>>> Is there any idea to improve this proceeding?
>> What about this:
>>  1. select all items in ROI manager
>>  2. ROI Manager More>>Combine (may be slow with many particles)
>>  3. Create Mask
>>  4. correct whatever you like (draw, delete, fill)
>>  5. run "Analyze Particles" on the mask
>>  6. delete everything in the ROI Manager
>>  7. ROI Manager More>>Add Particles
>>
>> 1.-3. is not needed in case you have the mask of all objects already.
> Thank you, I tried that. This way is similar to applying analyze
> particle... with appropriate options after mask correction.
>
> Regards
> Karsten
>
> ------------------------------
>
> Date:    Mon, 9 Jul 2007 19:07:22 -0700
> From:    Jon Harman <[hidden email]>
> Subject: ImageJ undo notification
>
> Hi,
>
> Just wondering if there is any way my plugin can find out if undo has
> been done on an image.  I have set up my plugin to implement ImageJ's
> undo command and undo works within my plugin.  My plugin can do many
> different things to an image.  For the output result I create a name
> that reflects what has been done to the image. So I would like to know
> if the user has undone a step.
>
> More generally I'd like to know the sequence of ImageJ commands that
> have been performed on an image.  Does ImageJ keep a log of this
> somewhere?  It must have some capability to do this in the macro
> recorder.  My goal will be to document exactly what has been done to an
> input image to get the output image.  I can keep track of  this for my
> own plugin commands, but would like to also keep track of any ImageJ
> commands (smoothing, sharpen, etc) that have been done to the image.
>
> Thanks,
> Jon
>
> ------------------------------
>
> End of IMAGEJ Digest - 8 Jul 2007 to 9 Jul 2007 (#2007-178)
> ***********************************************************
>

[Herbert M. Geller]

Here is a link that might help you:
http://dir.nhlbi.nih.gov/labs/ldn/comet.asp

Hélio Miot wrote:

> Hi all,
> I'm having some difficulties to
> estimate the tail length from comet assay.
> And can't segmentate the nucleus alone.
>
> Is there any plugin?
>
> Thanks,
>
> Hélio A. Miot
> Professor Assistente Doutor
> Departamento de Dermatologia, S/N
> Faculdade de Medicina da UNESP
> Campus Universitário de Rubião Jr.
> 18618-000   -   Botucatu - SP

--
--------------------------------------
Herbert M. Geller, Ph.D.
Developmental Neurobiology Section
National Heart Lung and Blood Institute, NIH
10 Center Drive MSC 1754
Bldg 10, Room 6D18
Bethesda, MD  20892-1754
Tel: [phone]; Fax: [phone]
e-mail: [hidden email]
Web: http://dir.nhlbi.nih.gov/labs/ldn/index.asp
---------------------------------------

[Ales Kladnik]

in our lab we have written a manual for doing comet-assay:
http://botanika.biologija.org/exp/comet/

Ales Kladnik

H said...
> I'm having some difficulties to
> estimate the tail length from comet assay.
> And can't segmentate the nucleus alone.
>
> Is there any plugin?
>
> Thanks,
>
> Hélio A. Miot


--
Ales Kladnik
University of Ljubljana, Biotechnical Faculty, Department of Biology
Vecna pot 111, SI-1000 Ljubljana, Slovenia
tel: [phone], fax: [phone]
url: http://botanika.biologija.org/
skype: fridjo, msn: [hidden email]