Quantifying Immunocytochemistry using Fluorescent Antibodies
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Dear all~~
I am relatively new to ImageJ, and have spent many hours trying to find the answer to my questions about quantifying the fluorescence of tagged antibodies, but to no avail. I am working with cell cultures, look at specific proteins tagged with fluorescent antibody, so there is a tendency for the fluorescence to have high background. Are there any plugins currently available that subtract out the background, such that only the strongest signals (above a certain threshold) are seen in a biologically relevant fashion? Do any of the colocalization plugins do this? I am trying to use the tools that are currently available, but with poor results, which I assume is partially due to my inexperience with ImageJ. I am interested in then counting the number of pixel clusters, and analyzing their intensities. Any information would be very much appreciated. Thank you! ~tatiana |
Re: Quantifying Immunocytochemistry using Fluorescent Antibodies
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Hi Tatiana,
The ShapeLogic plugin has a Color Particle Analyzer with menu names: * ColorParticleAnalyzer * RGBColorParticleAnalyzer ColorParticleAnalyzer will try to find background and then segment out the pixel clusters and list their intensities and other geometric features. For the RGBColorParticleAnalyzer you can set the background yourself and a max distance. If you just want to see the image after cutting the background off at a given level, you can use the ColorReplacer. -Sami Badawi http://www.shapelogic.org On Fri, Apr 10, 2009 at 7:00 PM, tatiana <[hidden email]> wrote: > Dear all~~ > > I am relatively new to ImageJ, and have spent many hours trying to find the answer to my questions about quantifying the fluorescence of tagged antibodies, but to no avail. I am working with cell cultures, look at specific proteins tagged with fluorescent antibody, so there is a tendency for the fluorescence to have high background. Are there any plugins currently available that subtract out the background, such that only the strongest signals (above a certain threshold) are seen in a biologically relevant fashion? Do any of the colocalization plugins do this? I am trying to use the tools that are currently available, but with poor results, which I assume is partially due to my inexperience with ImageJ. I am interested in then counting the number of pixel clusters, and analyzing their intensities. > > Any information would be very much appreciated. > > Thank you! > ~tatiana > -- > View this message in context: http://n2.nabble.com/Quantifying-Immunocytochemistry-using-Fluorescent-Antibodies-tp2618764p2618764.html > Sent from the ImageJ mailing list archive at Nabble.com. > |
Re: Quantifying Immunocytochemistry using Fluorescent Antibodies
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In reply to this post by tatiana
Tatiana,
It is not clear what you are doing and what the problem is. You mention quantifying and also colocalization tools - different things and quantifying colocalization comes with it's own bag of worms. I will assume that quantifying brightness of a given detail between 2 images using the same probe (stain/antibody/label) and the sample is suspected of showing different degrees of probe binding in the conditions compared. It is always best to do everything possible to reduce the background first so that processing is not needed. Also, please be aware of the ethical issues around processing of images. Many journals require disclose of all steps used. I have 2 links on our web page to some information sources on this topic. http://www.bio.umass.edu/microscopy/resource.htm Having said that, lets talk processing.... I will assume you are using a scientific camera that produces a monochrome image for each channel and not a consumer RGB image producer. [[Are you collecting a monochrome image (from a monochrome camera, one channel of fluorescence in the image) or a color image using a consumer-type camera that produces an RGB image (even if just in one channel ("cube") of your microscope? If it is this case you should convert the image type to "8-bit" to make it a monochrome image (grayscale image = intensity of that channel). Note that some filter sets that use longpass (LP) emission filters will show extended color - for example a blue excitation, 500 dichroic and a 525LP used on leaf tissue with GFP and intrinsic chlorophyll will let you visualize GFP but will also show red chloroplasts - and a green bandpass would be a better choice - since the red is not really "background". You should use a filter that best isolates your intended signal.]] If you are quantifying results between images you need to be very careful with the processing; the same exposure should be used for each image with no other changes in any of the microscope or collection parameters, and this includes stable illumination. Set the exposure to give approximately 0.9 full-scale value with the sample that gives the brightest image (to avoid saturation), and take the other image to be compared with the same exposure, so the maximum value in the second image should always be less than or equal to the control. A 12-bit camera is good for this type of work. Assuming you have grayscale images of a single channel and there is simply a diffuse brightness of the whole field and your specimen is there and somewhat more intense (and suspected to be different in brightness between the 2 samples/images), open the images and use "Analyze|Histogram" to get image data; the MIN value is of interest here, and you need to use the value reported for the image with the LOWEST background... Use the "Process|Math" tool to do the operations, in this case to enter a value to be subtracted from each pixel in each the image. Use the MIN value returned by the Histogram function from the image with a lower background - the same value should be applied to both images to be compared. Note: If there is still a background remaining in one of the images after these operations, there is no basis for removing it, sorry; to do so may make the image look nicer, but quantitative work is then out the window. [For "visual-only" quick cleanup, or exploring these operations graphically, choose "Process|Subtract Background" and go with the defaults and the value it found as an average base offset from what it found for the "detail" will be subtracted. This same process occurs in the "Process|Enhance Contrast" selection except in that method the background subtracted image gets every pixel subsequently multiplied by a value to make the remaining brightest pixel reach 255, and of course other pixels will be in proportion, but this is not good for quantification....] If two images are taken with the same setup, same exposure, have a constant background subtracted from each, and optionally, a same "stretch" value applied to each, then the values found by measuring over features (cells, etc.) can be compared. Hope this helps. Dale tatiana wrote: > ---------------------- Information from the mail header ----------------------- > Sender: ImageJ Interest Group <[hidden email]> > Poster: tatiana <[hidden email]> > Subject: Quantifying Immunocytochemistry using Fluorescent Antibodies > ------------------------------------------------------------------------------- > > Dear all~~ > > I am relatively new to ImageJ, and have spent many hours trying to find the answer to my questions about quantifying the fluorescence of tagged antibodies, but to no avail. I am working with cell cultures, look at specific proteins tagged with fluorescent antibody, so there is a tendency for the fluorescence to have high background. Are there any plugins currently available that subtract out the background, such that only the strongest signals (above a certain threshold) are seen in a biologically relevant fashion? Do any of the colocalization plugins do this? I am trying to use the tools that are currently available, but with poor results, which I assume is partially due to my inexperience with ImageJ. I am interested in then counting the number of pixel clusters, and analyzing their intensities. > > Any information would be very much appreciated. > > Thank you! > ~tatiana |
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In reply to this post by tatiana
Hi all~~
Thank you for your replies! Apologies for being unclear in my initial description. I am taking these images on a spinning disk confocal microscopy in grey scale, so yes, with a scientific camera. Dissociated cell cultures are either incubated with an antibody to a specific protein (standard immunocytochemistry), or transfected with a GFP tagged plasmid. I am trying to quantify the protein antibody (both in number and intensity), but am having difficulty with the background noise. I am attempting your suggestions in subtracting out the background, and then quantifying. I agree with you on the potential biases that can come with image processing, which is why I would prefer not to use any processing if possible. I guess my initial questions came from how to subtract out the background in a reasonable way. Thanks again for your help! If there are still some questions about above, please let me know. Regards, ~tatiana
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Re: Quantifying Immunocytochemistry using Fluorescent Antibodies
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Have you tried doing deconvolution on your images?
I have never done this using imageJ so wouldnt be able to recommend a plugin as I use a deltavision RT microscope which comes with its own deconvolution software for putting out of focus light back in focus. Deconvolution can help massively at removing high background caused by diffuse non-specific staining/signal. For example... when staining actin you can get high background which is almost completely removed by deconvolution. For other things deconvolution has hardly any effect at all and can make things worse. I find this is the case for certain GFP fusion proteins that have low signal intensity. might be worth trying deconvolution though ----- Original Message ----- From: "tatiana" <[hidden email]> To: <[hidden email]> Sent: Tuesday, April 14, 2009 8:42 PM Subject: Re: Quantifying Immunocytochemistry using Fluorescent Antibodies > Hi all~~ > > Thank you for your replies! Apologies for being unclear in my initial > description. > > I am taking these images on a spinning disk confocal microscopy in grey > scale, so yes, with a scientific camera. Dissociated cell cultures are > either incubated with an antibody to a specific protein (standard > immunocytochemistry), or transfected with a GFP tagged plasmid. I am > trying to quantify the protein antibody (both in number and intensity), > but am having difficulty with the background noise. I am attempting your > suggestions in subtracting out the background, and then quantifying. I > agree with you on the potential biases that can come with image > processing, which is why I would prefer not to use any processing if > possible. I guess my initial questions came from how to subtract out the > background in a reasonable way. > > Thanks again for your help! If there are still some questions about > above, please let me know. > > Regards, > ~tatiana > > > > Dear all~~ > > I am relatively new to ImageJ, and have spent many hours trying to find > the answer to my questions about quantifying the fluorescence of tagged > antibodies, but to no avail. I am working with cell cultures, look at > specific proteins tagged with fluorescent antibody, so there is a tendency > for the fluorescence to have high background. Are there any plugins > currently available that subtract out the background, such that only the > strongest signals (above a certain threshold) are seen in a biologically > relevant fashion? Do any of the colocalization plugins do this? I am > trying to use the tools that are currently available, but with poor > results, which I assume is partially due to my inexperience with ImageJ. > I am interested in then counting the number of pixel clusters, and > analyzing their intensities. > > Any information would be very much appreciated. > > Thank you! > ~tatiana > > > -- > View this message in context: > http://n2.nabble.com/Quantifying-Immunocytochemistry-using-Fluorescent-Antibodies-tp2618764p2634951.html > Sent from the ImageJ mailing list archive at Nabble.com. > |
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There a plugin for obtain Psf Fitting for astronomical images?
Thanks, Gabriele |
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In reply to this post by Michael Cundell
I think at stellar psf, a function of Moffat or simplement a gaussian
fitting. I need to know the exact center of a spot light. Reference is daophot of P.Stetson. Thanks [hidden email] ha scritto: Hi Gabrielle, I do not know about a current plugin, but would be interested if there is one. Actually I have been working in this field for quite a while, even if we are at the other end of the size scale, we do "PSF fitting" of microscopic images. |
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In reply to this post by Gabriele Umbriaco
Hi Gabriele,
One group within the French Technological Network for Microscopies (rough translation, but have a look at http://rtmfm.ibl.fr/) is currently working on a package to check the health of the microscopes. This package, called MetroloJ (still experimental, not yet released) includes a PSF fitting plugin, aimed at determination of the microscope's resolution, based on PSF profiles analysis. This plugin might be tweaked a bit in order to fulfill your needs. If you're interested, I might send it to you offlist. Fabrice Le 15 avr. 09 à 11:25, gabriele umbriaco a écrit : > There a plugin for obtain Psf Fitting for astronomical images? > Thanks, Gabriele > Fabrice Cordelières, PhD Institut Curie - Section de recherche/ CNRS UMR 146 Plateforme d'Imagerie Cellulaire et Tissulaire Bâtiment 112 - Centre universitaire 91405 Orsay Cedex FRANCE Tél. : +33 1 69 86 31 30 Fax. : +33 1 69 86 17 03 Consultez mes disponibilités sur http://www.google.com/calendar/embed?src=fabrice.cordelieres%40gmail.com |
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Yes, I try it.
Thanks Fabrice Cordelières ha scritto: > Hi Gabriele, > > One group within the French Technological Network for Microscopies > (rough translation, but have a look at http://rtmfm.ibl.fr/) is > currently working on a package to check the health of the microscopes. > This package, called MetroloJ (still experimental, not yet released) > includes a PSF fitting plugin, aimed at determination of the > microscope's resolution, based on PSF profiles analysis. This plugin > might be tweaked a bit in order to fulfill your needs. If you're > interested, I might send it to you offlist. > > Fabrice > > > > Le 15 avr. 09 à 11:25, gabriele umbriaco a écrit : > >> There a plugin for obtain Psf Fitting for astronomical images? >> Thanks, Gabriele >> > > > Fabrice Cordelières, PhD > > Institut Curie - Section de recherche/ CNRS UMR 146 > Plateforme d'Imagerie Cellulaire et Tissulaire > Bâtiment 112 - Centre universitaire > 91405 Orsay Cedex > FRANCE > > Tél. : +33 1 69 86 31 30 > Fax. : +33 1 69 86 17 03 > > Consultez mes disponibilités sur > http://www.google.com/calendar/embed?src=fabrice.cordelieres%40gmail.com > |
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In reply to this post by Fabrice Cordelières
Hi Fabrice,
as I wrote (by error it did not go directly to the list, but is included in Gabrielle´s 1st reply) I would be very much interested too in any such stuff and would like to try the plugin! Mit freundlichen Grüßen / Best regards Dr. Joachim Wesner Projektleiter Optik Technologiesysteme ____________________________________________ Leica Microsystems CMS GmbH | GmbH mit Sitz in Wetzlar | Amtsgericht Wetzlar HRB 2432 Geschäftsführer: Dr. Stefan Traeger | Dr. Wolf-Otto Reuter | Dr. David Roy Martyr | Colin Davis Fabrice Cordelières <fabrice.cordelie An [hidden email]-PSUD. [hidden email] FR> Kopie Gesendet von: ImageJ Interest Thema Group Re: PSF FITTING <[hidden email]. GOV> 15.04.2009 13:55 Bitte antworten an ImageJ Interest Group <[hidden email]. GOV> Hi Gabriele, One group within the French Technological Network for Microscopies (rough translation, but have a look at http://rtmfm.ibl.fr/) is currently working on a package to check the health of the microscopes. This package, called MetroloJ (still experimental, not yet released) includes a PSF fitting plugin, aimed at determination of the microscope's resolution, based on PSF profiles analysis. This plugin might be tweaked a bit in order to fulfill your needs. If you're interested, I might send it to you offlist. Fabrice Le 15 avr. 09 à 11:25, gabriele umbriaco a écrit : > There a plugin for obtain Psf Fitting for astronomical images? > Thanks, Gabriele > Fabrice Cordelières, PhD Institut Curie - Section de recherche/ CNRS UMR 146 Plateforme d'Imagerie Cellulaire et Tissulaire Bâtiment 112 - Centre universitaire 91405 Orsay Cedex FRANCE Tél. : +33 1 69 86 31 30 Fax. : +33 1 69 86 17 03 Consultez mes disponibilités sur http://www.google.com/calendar/embed?src=fabrice.cordelieres%40gmail.com ______________________________________________________________________ This email has been scanned by the MessageLabs Email Security System. For more information please visit http://www.messagelabs.com/email ______________________________________________________________________ |
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Don't forget my Plugin. It measures FWHM of points of light, and can
find them too if they are on a sufficiently contrasty background (such as deep space). We use it for measuring detector resolution too. It does 2d gaussian fits using the Levenburg-Marquardt method. And I wrote some decent documentation for it too... http://rsb.info.nih.gov/ij/plugins/fwhm/index.html Dr Adrian Martin Sensor Sciences, 3333 Vincent Road, Suite 103, Pleasant Hill, CA 94523 (925) 296 0848 phone, (925) 296 0849 fax On Apr 15, 2009, at 5:47 AM, Joachim Wesner wrote: > Hi Fabrice, > > as I wrote (by error it did not go directly to the list, but is > included in > Gabrielle´s 1st reply) > I would be very much interested too in any such stuff and would like > to try > the plugin! > > > Mit freundlichen Grüßen / Best regards > > Dr. Joachim Wesner > Projektleiter Optik Technologiesysteme > > ____________________________________________ > > Leica Microsystems CMS GmbH | GmbH mit Sitz in Wetzlar | Amtsgericht > Wetzlar HRB 2432 > Geschäftsführer: Dr. Stefan Traeger | Dr. Wolf-Otto Reuter | Dr. > David Roy > Martyr | Colin Davis > > > > Fabrice > Cordelières > > <fabrice.cordelie An > [hidden email]-PSUD. [hidden email] > FR> > Kopie > Gesendet von: > ImageJ Interest > Thema > Group Re: PSF FITTING > <[hidden email]. > GOV> > > > 15.04.2009 13:55 > > > Bitte antworten > an > ImageJ Interest > Group > <[hidden email]. > GOV> > > > > > > > Hi Gabriele, > > One group within the French Technological Network for Microscopies > (rough translation, but have a look at http://rtmfm.ibl.fr/) is > currently working on a package to check the health of the microscopes. > This package, called MetroloJ (still experimental, not yet released) > includes a PSF fitting plugin, aimed at determination of the > microscope's resolution, based on PSF profiles analysis. This plugin > might be tweaked a bit in order to fulfill your needs. If you're > interested, I might send it to you offlist. > > Fabrice > > > > Le 15 avr. 09 à 11:25, gabriele umbriaco a écrit : > >> There a plugin for obtain Psf Fitting for astronomical images? >> Thanks, Gabriele >> > > > Fabrice Cordelières, PhD > > Institut Curie - Section de recherche/ CNRS UMR 146 > Plateforme d'Imagerie Cellulaire et Tissulaire > Bâtiment 112 - Centre universitaire > 91405 Orsay Cedex > FRANCE > > Tél. : +33 1 69 86 31 30 > Fax. : +33 1 69 86 17 03 > > Consultez mes disponibilités sur > http://www.google.com/calendar/embed?src=fabrice.cordelieres%40gmail.com > > > ______________________________________________________________________ > This email has been scanned by the MessageLabs Email Security System. > For more information please visit http://www.messagelabs.com/email > ______________________________________________________________________ |
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