how to measure
Locked
 
|
Hello,
because I am quite unexperienced in measuring fluorescent signals I hope somebody may give me some fruitful instructions for doing with imageJ. Transfected HEK cells (expressing a presynaptic protein -green channel) were cocultured with hippocampal cells. I wish to measure the signals of a postsynaptic proteins (red channel) on transfected HEK cells. In other words: I am interested to measure the overlap between the red an the green channel. My idea was to mark the green signal (transfected HEK cells) and to measure the intensities in the red channel. I did it that way: 1) Fusing stacks (Image-Hyperstack-Reduce Dimensionality) 2) Make it possible to switch between channels (Image-Hyperstack-Channels) 3) Mark the green signal with the freehand tool and then switch to the red channel. 4) Measure the intensities of the red signal within the marked area (Analyze-Measure). I feel there is a better more efficient way to do. Is there? Especially the method to define the region of interest (the overlap) could be more precise, couldn´t it... Thanks Johannes |
|
|
It looks to me as if you should look at some of the colocalization
plugins. These allow you to select just those cells that share both labels, as long as the labels are in the same part of the cell. > Hello, > > because I am quite unexperienced in measuring fluorescent signals I hope > somebody may give me some fruitful instructions for doing with imageJ. > > Transfected HEK cells (expressing a presynaptic protein -green channel) were > cocultured with hippocampal cells. I wish to measure the signals of a > postsynaptic proteins (red channel) on transfected HEK cells. In other > words: I am interested to measure the overlap between the red an the green > channel. > > My idea was to mark the green signal (transfected HEK cells) and to measure > the intensities in the red channel. > > I did it that way: > 1) Fusing stacks (Image-Hyperstack-Reduce Dimensionality) > 2) Make it possible to switch between channels (Image-Hyperstack-Channels) > 3) Mark the green signal with the freehand tool and then switch to the red > channel. > 4) Measure the intensities of the red signal within the marked area > (Analyze-Measure). > > I feel there is a better more efficient way to do. Is there? Especially the > method to define the region of interest (the overlap) could be more precise, > couldn´t it... > > Thanks Johannes -- Joel B. Sheffield, Ph.D. Biology Department, Temple University 1900 North 12th Street Philadelphia, PA 19122 [hidden email] (215) 204 8839, fax (215) 204 0486 http://astro.temple.edu/~jbs |
|
|
Johannes
I also believe you need to do colocalization analysis. Try JACoP (Just Another Co-localization Plugin) plug from Fabrice P. Cordelières. Here goes the site: http://rsbweb.nih.gov/ij/plugins/track/jacop.html Ruy Jaeger =============================================================================== Ruy G. Jaeger, DDS, MSD, PhD University of Sao Paulo - USP Institute of Biomedical Sciences - ICB Department of Cell and Developmental Biology Av. Prof. Lineu Prestes 1524 Ed Biomedicas 1 rooms 302 (office and microscopy room) and 405(Laboratory) Sao Paulo SP 05508 000 BRAZIL Phones:55-11-30918454(office), 55-1130918034 (Lab);Fax:55-11-30917402 email:[hidden email] website: http://www.icb.usp.br/~rgjaeger/index.html =============================================================================== Citando Joel Sheffield <[hidden email]>: > It looks to me as if you should look at some of the colocalization > plugins. These allow you to select just those cells that share both > labels, as long as the labels are in the same part of the cell. > > > >> Hello, >> >> because I am quite unexperienced in measuring fluorescent signals I hope >> somebody may give me some fruitful instructions for doing with imageJ. >> >> Transfected HEK cells (expressing a presynaptic protein -green channel) were >> cocultured with hippocampal cells. I wish to measure the signals of a >> postsynaptic proteins (red channel) on transfected HEK cells. In other >> words: I am interested to measure the overlap between the red an the green >> channel. >> >> My idea was to mark the green signal (transfected HEK cells) and to measure >> the intensities in the red channel. >> >> I did it that way: >> 1) Fusing stacks (Image-Hyperstack-Reduce Dimensionality) >> 2) Make it possible to switch between channels (Image-Hyperstack-Channels) >> 3) Mark the green signal with the freehand tool and then switch to the red >> channel. >> 4) Measure the intensities of the red signal within the marked area >> (Analyze-Measure). >> >> I feel there is a better more efficient way to do. Is there? Especially the >> method to define the region of interest (the overlap) could be more precise, >> couldn´t it... >> >> Thanks Johannes > > > -- > Joel B. Sheffield, Ph.D. > Biology Department, Temple University > 1900 North 12th Street > Philadelphia, PA 19122 > [hidden email] > (215) 204 8839, fax (215) 204 0486 > http://astro.temple.edu/~jbs > |
|
|
I tried some of the recommended plugins (under those JACoP seems to be the
most intuitive) but I never found the right subtool... I am not interested in measuring the degree of colocalization. I just want to measure fluorescent intensities of the red channel while I defined the ROI in the green channel. Are the colocalization plugins really the right tools? 2008/7/9 Ruy G. Jaeger <[hidden email]>: > Johannes > > I also believe you need to do colocalization analysis. Try JACoP (Just > Another Co-localization Plugin) plug from Fabrice P. Cordelières. Here goes > the site: > > http://rsbweb.nih.gov/ij/plugins/track/jacop.html > > Ruy Jaeger > > > =============================================================================== > Ruy G. Jaeger, DDS, MSD, PhD > University of Sao Paulo - USP Institute of Biomedical Sciences - ICB > Department of Cell and Developmental Biology > Av. Prof. Lineu Prestes 1524 > Ed Biomedicas 1 rooms 302 (office and microscopy room) and > 405(Laboratory) > Sao Paulo SP 05508 000 BRAZIL > Phones:55-11-30918454(office), 55-1130918034 (Lab);Fax:55-11-30917402 > email:[hidden email] <email%[hidden email]> website: > http://www.icb.usp.br/~rgjaeger/index.html<http://www.icb.usp.br/%7Ergjaeger/index.html> > > =============================================================================== > > > > Citando Joel Sheffield <[hidden email]>: > > > It looks to me as if you should look at some of the colocalization >> plugins. These allow you to select just those cells that share both >> labels, as long as the labels are in the same part of the cell. >> >> >> >> Hello, >>> >>> because I am quite unexperienced in measuring fluorescent signals I hope >>> somebody may give me some fruitful instructions for doing with imageJ. >>> >>> Transfected HEK cells (expressing a presynaptic protein -green channel) >>> were >>> cocultured with hippocampal cells. I wish to measure the signals of a >>> postsynaptic proteins (red channel) on transfected HEK cells. In other >>> words: I am interested to measure the overlap between the red an the >>> green >>> channel. >>> >>> My idea was to mark the green signal (transfected HEK cells) and to >>> measure >>> the intensities in the red channel. >>> >>> I did it that way: >>> 1) Fusing stacks (Image-Hyperstack-Reduce Dimensionality) >>> 2) Make it possible to switch between channels >>> (Image-Hyperstack-Channels) >>> 3) Mark the green signal with the freehand tool and then switch to the >>> red >>> channel. >>> 4) Measure the intensities of the red signal within the marked area >>> (Analyze-Measure). >>> >>> I feel there is a better more efficient way to do. Is there? Especially >>> the >>> method to define the region of interest (the overlap) could be more >>> precise, >>> couldn´t it... >>> >>> Thanks Johannes >>> >> >> >> -- >> Joel B. Sheffield, Ph.D. >> Biology Department, Temple University >> 1900 North 12th Street >> Philadelphia, PA 19122 >> [hidden email] >> (215) 204 8839, fax (215) 204 0486 >> http://astro.temple.edu/~jbs <http://astro.temple.edu/%7Ejbs> >> >> |
|
|
Johannes Breu a écrit :
> I tried some of the recommended plugins (under those JACoP seems to be the > most intuitive) but I never found the right subtool... I am not interested > in measuring the degree of colocalization. I just want to measure > fluorescent intensities of the red channel while I defined the ROI in the > green channel. Are the colocalization plugins really the right tools? > > 2008/7/9 Ruy G. Jaeger <[hidden email]>: > > >> Johannes >> >> I also believe you need to do colocalization analysis. Try JACoP (Just >> Another Co-localization Plugin) plug from Fabrice P. Cordelières. Here goes >> the site: >> >> http://rsbweb.nih.gov/ij/plugins/track/jacop.html >> >> Ruy Jaeger >> >> >> =============================================================================== >> Ruy G. Jaeger, DDS, MSD, PhD >> University of Sao Paulo - USP Institute of Biomedical Sciences - ICB >> Department of Cell and Developmental Biology >> Av. Prof. Lineu Prestes 1524 >> Ed Biomedicas 1 rooms 302 (office and microscopy room) and >> 405(Laboratory) >> Sao Paulo SP 05508 000 BRAZIL >> Phones:55-11-30918454(office), 55-1130918034 (Lab);Fax:55-11-30917402 >> email:[hidden email] <email%[hidden email]> website: >> http://www.icb.usp.br/~rgjaeger/index.html<http://www.icb.usp.br/%7Ergjaeger/index.html> >> >> =============================================================================== >> >> >> >> Citando Joel Sheffield <[hidden email]>: >> >> >> It looks to me as if you should look at some of the colocalization >> >>> plugins. These allow you to select just those cells that share both >>> labels, as long as the labels are in the same part of the cell. >>> >>> >>> >>> Hello, >>> >>>> because I am quite unexperienced in measuring fluorescent signals I hope >>>> somebody may give me some fruitful instructions for doing with imageJ. >>>> >>>> Transfected HEK cells (expressing a presynaptic protein -green channel) >>>> were >>>> cocultured with hippocampal cells. I wish to measure the signals of a >>>> postsynaptic proteins (red channel) on transfected HEK cells. In other >>>> words: I am interested to measure the overlap between the red an the >>>> green >>>> channel. >>>> >>>> My idea was to mark the green signal (transfected HEK cells) and to >>>> measure >>>> the intensities in the red channel. >>>> >>>> I did it that way: >>>> 1) Fusing stacks (Image-Hyperstack-Reduce Dimensionality) >>>> 2) Make it possible to switch between channels >>>> (Image-Hyperstack-Channels) >>>> 3) Mark the green signal with the freehand tool and then switch to the >>>> red >>>> channel. >>>> 4) Measure the intensities of the red signal within the marked area >>>> (Analyze-Measure). >>>> >>>> I feel there is a better more efficient way to do. Is there? Especially >>>> the >>>> method to define the region of interest (the overlap) could be more >>>> precise, >>>> couldn´t it... >>>> >>>> Thanks Johannes >>>> >>>> >>> -- >>> Joel B. Sheffield, Ph.D. >>> Biology Department, Temple University >>> 1900 North 12th Street >>> Philadelphia, PA 19122 >>> [hidden email] >>> (215) 204 8839, fax (215) 204 0486 >>> http://astro.temple.edu/~jbs <http://astro.temple.edu/%7Ejbs> >>> >>> >>> I would try to do segmentation on the green channel, use analyze particules to define the ROI, add those ROI to the ROI-manager, use that set of ROI to measure in the red channel. Cheers, Fabrice. -- Senger Fabrice |
|
|
In reply to this post by Johannes Breu
---- Original message ----
>Date: Thu, 10 Jul 2008 08:46:21 +0200 >From: Senger Fabrice <[hidden email]> >Subject: Re: how to measure >To: [hidden email] > >Johannes Breu a écrit : >> I tried some of the recommended plugins (under those JACoP seems to be the >> most intuitive) but I never found the right subtool... I am not interested >> in measuring the degree of colocalization. I just want to measure >> fluorescent intensities of the red channel while I defined the ROI in the >> green channel. Are the colocalization plugins really the right tools? >> >>>> >>>> >Hi there, > >I would try to do segmentation on the green channel, use analyze >particules to define the ROI, add those ROI to the ROI-manager, >use that set of ROI to measure in the red channel. > >Cheers, > > >Fabrice. > >-- >Senger Fabrice Alternatively, you could threshold and make a mask of your green image, then use the Edit --> Selection --> Create Selection tool to store your ROI. Threshold and make binary your red image and Edit --> Selection --> Restore selection. Then analyze particles will run only within the selected area. I guess it depends on how you want to define your ROI. selectWindow("green_image"); run("Threshold..."); run("Convert to Mask"); run("Create Selection"); selectWindow("red_image"); run("Threshold..."); run("Restore Selection"); run("Analyze Particles...",); Christine Christine Labno, Ph.D. Asst. Technical Director Light Microscopy Core University of Chicago Office of Shared Research Facilities Abbott 129 (773) 834-9040 (phone) |
|
|
2008/7/10 Christine Labno <[hidden email]>:
> ---- Original message ---- > >Date: Thu, 10 Jul 2008 08:46:21 +0200 > >From: Senger Fabrice <[hidden email]> > >Subject: Re: how to measure > >To: [hidden email] > > > >Johannes Breu a écrit : > >> I tried some of the recommended plugins (under those JACoP > seems to be the > >> most intuitive) but I never found the right subtool... I am > not interested > >> in measuring the degree of colocalization. I just want to > measure > >> fluorescent intensities of the red channel while I defined > the ROI in the > >> green channel. Are the colocalization plugins really the > right tools? > >> > > >>>> > >>>> > >Hi there, > > > >I would try to do segmentation on the green channel, use analyze > >particules to define the ROI, add those ROI to the ROI-manager, > >use that set of ROI to measure in the red channel. > > > >Cheers, > > > > > >Fabrice. > > > >-- > >Senger Fabrice > > > Alternatively, you could threshold and make a mask of your > green image, then use the Edit --> Selection --> Create > Selection tool to store your ROI. Threshold and make binary > your red image and Edit --> Selection --> Restore selection. > Then analyze particles will run only within the selected area. > I guess it depends on how you want to define your ROI. > > selectWindow("green_image"); > run("Threshold..."); > run("Convert to Mask"); > run("Create Selection"); > selectWindow("red_image"); > run("Threshold..."); > run("Restore Selection"); > run("Analyze Particles...",); > > > Christine > > > Christine Labno, Ph.D. > Asst. Technical Director > Light Microscopy Core > University of Chicago > Office of Shared Research Facilities > Abbott 129 > (773) 834-9040 (phone) > Thank you very much. Both methods seem to work. I have to figure out what´s best for my purpose. The RestoreSelection method has the advantage that I get only one result line for each image after measuring. This seems to be more managable. There are still some questions: 1) It seems to be standard to analyze 8bit images. Why? I thougth it was better to perform the analysis using 16 bit images because there is more information. Is that true? 2) I prefer the Analyze-Measure (instead of Analyze-Analyze Particles) for measuring the red channel (the dots I am interested within the ROI). But I would be more happy if I gain information about the size of the area just measured. Where do I find it? (Set Measurements? the output for integrated density was zero). What is the advantage of ANALYZE PARTICLES in this context? 3) I have a principle problem working with threshold. As I have to compare a lot of images that I do have to treat similarily I have the impression it is better to use SUBTRACT. Could you please comment on this. Thank you very much Johannes |
|
|
Johannes Breu a écrit :
> 2008/7/10 Christine Labno <[hidden email]>: > > >> ---- Original message ---- >> >>> Date: Thu, 10 Jul 2008 08:46:21 +0200 >>> From: Senger Fabrice <[hidden email]> >>> Subject: Re: how to measure >>> To: [hidden email] >>> >>> Johannes Breu a écrit : >>> >>>> I tried some of the recommended plugins (under those JACoP >>>> >> seems to be the >> >>>> most intuitive) but I never found the right subtool... I am >>>> >> not interested >> >>>> in measuring the degree of colocalization. I just want to >>>> >> measure >> >>>> fluorescent intensities of the red channel while I defined >>>> >> the ROI in the >> >>>> green channel. Are the colocalization plugins really the >>>> >> right tools? >> >>>>>> >>> Hi there, >>> >>> I would try to do segmentation on the green channel, use analyze >>> particules to define the ROI, add those ROI to the ROI-manager, >>> use that set of ROI to measure in the red channel. >>> >>> Cheers, >>> >>> >>> Fabrice. >>> >>> -- >>> Senger Fabrice >>> >> Alternatively, you could threshold and make a mask of your >> green image, then use the Edit --> Selection --> Create >> Selection tool to store your ROI. Threshold and make binary >> your red image and Edit --> Selection --> Restore selection. >> Then analyze particles will run only within the selected area. >> I guess it depends on how you want to define your ROI. >> >> selectWindow("green_image"); >> run("Threshold..."); >> run("Convert to Mask"); >> run("Create Selection"); >> selectWindow("red_image"); >> run("Threshold..."); >> run("Restore Selection"); >> run("Analyze Particles...",); >> >> >> Christine >> >> >> Christine Labno, Ph.D. >> Asst. Technical Director >> Light Microscopy Core >> University of Chicago >> Office of Shared Research Facilities >> Abbott 129 >> (773) 834-9040 (phone) >> >> > > Thank you very much. Both methods seem to work. I have to figure out what´s > best for my purpose. The RestoreSelection method has the advantage that I > get only one result line for each image after measuring. This seems to be > more managable. > > There are still some questions: > > 1) It seems to be standard to analyze 8bit images. Why? I thougth it was > better to perform the analysis using 16 bit images because there is more > information. Is that true? > Yes there is more info. you could create you set of ROI's and send them back to the original 16bit image for measurement. > 2) I prefer the Analyze-Measure (instead of Analyze-Analyze Particles) for > measuring the red channel (the dots I am interested within the ROI). But I > would be more happy if I gain information about the size of the area just > measured. Where do I find it? (Set Measurements? the output for integrated > density was zero). What is the advantage of ANALYZE PARTICLES in this > context? > Analyze particules lets you input information to select the ROI's, like size of particules, etc...of course, ones you have your set of ROI you will use Analyze-Measure, to get your info you should set measurements before runing measure. > 3) I have a principle problem working with threshold. As I have to compare a > lot of images that I do have to treat similarily I have the impression it is > better to use SUBTRACT. Could you please comment on this. > > To define your ROI's or to make a mask you have to binarize your image (at least a copy of it) and a binary image is basically thresholded. In my opinion, if your samples are prepared the same way, and the acquisitions are made in a systematic way, you might use the same threshold for all pictures. > Thank you very much > Johannes > -- Senger Fabrice |
|
|
In reply to this post by Ruy
Hello all,
I'm new to ImageJ and hope that someone can give me some advice on what I'd like to do. I'm trying to highlight and count colocalization of two types of antibody labeling. Both antibodies label many nuclei and I'm trying to identify the few that have colocalization. What I want to do is the following: 1. Identify 3D objects of the labeled nuclei for each antibody. (Channel 1 and 2) 2. Highlight all the colocalized pixels using the Colocalization Highlighter plugin. (Channel 3) 3. Creat 3D objects for the colocalized voxels. (Channel 3) 4. Compare the 3D objects in all three channels, filter out the objects that don't have the same (or within a certain threshold) volume in all three channels, then count what's left. I figured out how to do the first three tasks. I'm not sure what's the best way to deal with the fourth one. Thanks very much for your help! Adelaine Adelaine Leung Kravitz Lab Department of Neurobiology Harvard Medical School 220 Longwood Avenue Boston, MA 02115, USA Phone: 617-432-1042 Fax: 617-734-7557 |
Re: 3D object colocalization
|
|
Hi Adelaine,
you proposed method sounds logical, but will be prone to strong subjective bias. That is a bad thing. ...you need to look at the archives of this mailing list for posts from me and others about objective and quantitative colocalisation analysis, esp using the methods of costes et al and manders et al I will send you papers in another mail. once you have measured your colcoalisation between channels, you can make a colocalisation map between each channel, and asses point 4 from there... somehow. cheers Dan On Jul 17, 2008, at 6:00 AM, IMAGEJ automatic digest system wrote: > From: Adelaine Leung <[hidden email]> > Subject: 3D object colocalization > > Hello all, > > I'm new to ImageJ and hope that someone can give me some advice on > what I'd like to do. I'm trying to highlight and count > colocalization of two types of antibody labeling. Both antibodies > label many nuclei and I'm trying to identify the few that have > colocalization. What I want to do is the following: > > 1. Identify 3D objects of the labeled nuclei for each antibody. > (Channel 1 and 2) > 2. Highlight all the colocalized pixels using the Colocalization > Highlighter plugin. (Channel 3) > 3. Creat 3D objects for the colocalized voxels. (Channel 3) > 4. Compare the 3D objects in all three channels, filter out the > objects that don't have the same (or within a certain threshold) > volume in all three channels, then count what's left. > > I figured out how to do the first three tasks. I'm not sure what's > the best way to deal with the fourth one. > > Thanks very much for your help! > > Adelaine Dr. Daniel James White BSc. (Hons.) PhD Senior Microscopist / Image Processing and Analysis Light Microscopy Facility Max Planck Institute of Molecular Cell Biology and Genetics Pfotenhauerstrasse 108 01307 DRESDEN Germany New Mobile Number!!! +49 (0)15114966933 (German Mobile) +49 (0)351 210 2627 (Work phone at MPI-CBG) +49 (0)351 210 1078 (Fax MPI-CBG LMF) http://www.bioimagexd.net http://www.chalkie.org.uk [hidden email] ( [hidden email] ) |
«
Return to ImageJ
|
1 view|%1 views
| Free forum by Nabble | Edit this page |