Anchall ............ |
Hello all,
This query is regarding image analysis.I am lookin for some way I can quantify the following data. I have images with a protein A usually localised in ER and Golgi.But when I overexpress another protein B which kind of solubilises PROTEIN A then I see diffuse expression of protein A all over the cell basically in cytoplasm.Now what I want to find out is whether the protein is still present in ER & Golgi but it is not so distinguisable as now there is lots in cytoplasm too.Is there any way I can quantify this.. using IMAGE J as i mostly use only that... I hope some help in this regard.. Thanks Anchal |
Chris Tully |
Anchal,
Regardless of which software you are using, you need some way of isolating the ER and Golgi from the rest of the cell. You may want to consider fixing the cells and using a membrane stain for isolate the ER and Golgi based on shape, or you will need to express a third FP in one of the proteins that populates the ER and Golgi membranes. With a seperate channel it should be relatively easy to derive ROIs from the membrane channel for use in measuring the Protein A distribution in the main image. Now, this technique is only partially accurate. Since both the ER and Golgi are relatively small bodies that do not fill the entire vertical space of the cell you really need to look at this problem in 3D. You will need a lot of slices because you want to get as close to a cubic voxel as possible. I am sure there are some 3D tools for Image J but I would recommend that you also check out commercial software such as 3D Constructor (for Image-Pro Plus; http://www.mediacy.com), Volocity (http://www.improvision.com/products/volocity/), and Imaris (http://www.bitplane.com/). Chris Tully On Thu, Nov 13, 2008 at 10:37 PM, Anchall ............ <[hidden email]> wrote: Hello all, -- Chris Tully Microscopy and Image Analysis Expert [hidden email] 240-888-1021 http://www.linkedin.com/in/christully |
Gudrun Ihrke |
In reply to this post by Anchall ............
Dear Anchal,
You may want to consider to wash out the cytoplasmic pool of the protein by permeabilizing the cells with saponin before fixation. You can then compare the membrane-bound pools under your different experimental conditions more easily. This method worked for us very well [see Savage et al (2002). A digenic disorder of human insulin action. Nature Genetics 31: 379-384]. We observed such stark differences in our case that we did not need to quantify, but it would have been technically feasible. Gudrun On Nov 13, 2008, at 10:37 PM, Anchall ............ wrote: > Hello all, > This query is regarding image analysis.I am lookin for > some way I can quantify the following data. > I have images with a protein A usually localised in ER and > Golgi.But when I overexpress another protein B which kind of > solubilises PROTEIN A then I see diffuse expression of protein A > all over the cell basically in cytoplasm.Now what I want to find > out is whether the protein is still present in ER & Golgi but it is > not so distinguisable as now there is lots in cytoplasm too.Is > there any way I can quantify this.. using IMAGE J as i mostly use > only that... > I hope some help in this regard.. > Thanks > Anchal > ____________________________________________ Gudrun Ihrke, Ph.D., CIV Research Assistant Professor Department of Pharmacology (C2025) Uniformed Services University School of Medicine 4301 Jones Bridge Road Bethesda, MD 20814 (301) 295 3225 [hidden email] |
xavier Sanjuan |
Another option wolud be the use of digitonin instead of saponin. It
permeabilizes cholesterol-rich membranes (= plasma membrane but not intracellular organelle membranes) when used at appropriate concentrations. See Lorenz et al. Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization. Nature Methods - 3, 205 - 210 (2006). Best regards, Xavi. ___________________________________ Xavier Sanjuan Servei de Microscòpia Confocal Departament de Ciències Experimentals i de la Salut Universitat Pompeu Fabra Parc de Recerca Biomèdica de Barcelona Doctor Aiguader, 88 - Sala 309 08003 Barcelona - Spain Tel.: + 34 93 316 08 64 Fax: + 34 93 316 09 01 E-mail: [hidden email] Web: http://www.upf.edu/cexs/sct -----Mensaje original----- De: Confocal Microscopy List [mailto:[hidden email]] En nombre de Gudrun Ihrke Enviado el: sábado, 15 de noviembre de 2008 2:04 Para: [hidden email] Asunto: Re: image analysis-distribution of protein Dear Anchal, You may want to consider to wash out the cytoplasmic pool of the protein by permeabilizing the cells with saponin before fixation. You can then compare the membrane-bound pools under your different experimental conditions more easily. This method worked for us very well [see Savage et al (2002). A digenic disorder of human insulin action. Nature Genetics 31: 379-384]. We observed such stark differences in our case that we did not need to quantify, but it would have been technically feasible. Gudrun On Nov 13, 2008, at 10:37 PM, Anchall ............ wrote: > Hello all, > This query is regarding image analysis.I am lookin for > some way I can quantify the following data. > I have images with a protein A usually localised in ER and > Golgi.But when I overexpress another protein B which kind of > solubilises PROTEIN A then I see diffuse expression of protein A > all over the cell basically in cytoplasm.Now what I want to find > out is whether the protein is still present in ER & Golgi but it is > not so distinguisable as now there is lots in cytoplasm too.Is > there any way I can quantify this.. using IMAGE J as i mostly use > only that... > I hope some help in this regard.. > Thanks > Anchal > ____________________________________________ Gudrun Ihrke, Ph.D., CIV Research Assistant Professor Department of Pharmacology (C2025) Uniformed Services University School of Medicine 4301 Jones Bridge Road Bethesda, MD 20814 (301) 295 3225 [hidden email] |
In reply to this post by Chris Tully
Chris Tully wrote:
> Anchal, > > Regardless of which software you are using, you need some way of > isolating the ER and Golgi from the rest of the cell. You may want to > consider fixing the cells and using a membrane stain for isolate the > ER and Golgi based on shape, or you will need to express a third FP in > one of the proteins that populates the ER and Golgi membranes. With a > seperate channel it should be relatively easy to derive ROIs from the > membrane channel for use in measuring the Protein A distribution in > the main image. Now, this technique is only partially accurate. > Since both the ER and Golgi are relatively small bodies that do not > fill the entire vertical space of the cell you really need to look at > this problem in 3D. You will need a lot of slices because you want to > get as close to a cubic voxel as possible. > > I am sure there are some 3D tools for Image J but I would recommend > that you also check out commercial software such as 3D Constructor > (for Image-Pro Plus; http://www.mediacy.com <http://www.mediacy.com>), > Volocity (http://www.improvision.com/products/volocity/), and Imaris > (http://www.bitplane.com/). > /Johan -- -- ------------------------------------------------ Johan Henriksson MSc Engineering PhD student, Karolinska Institutet http://mahogny.areta.org http://www.endrov.net |
David Basiji |
***Commercial response***
Johan, et al., We do something similar to what you describe in terms of labeling the structure of interest and then using that image to determine an ROI for assessing localization or another label to that structure. I agree that the absolute error in doing this with a 2D projection of a single cell may be high due to orientation and focus considerations. However, it is not necessary to form a 3D image if you have the ability to measure enough individual cells in 2D, whether using our system or any other 2D imaging platform. Rather than relying on the localization measurement of a single cell, if you assess localization by measuring the distribution of measurements over many cells the assessment will be very robust. The error in each individual measurement will tend to broaden the distribution but since the odds of all the cumulative errors being in the "same direction" will be very low, the mean of the distribution will be very consistent. Best Regards, David David Basiji, Ph.D. President and CEO, Amnis Corporation 2505 Third Ave., Suite 210 Seattle, WA 98121 +1 206 374 7165 direct +1 206 919 3342 mobile +1 206 576 6895 fax www.amnis.com This email and any attachment contain information which is intended for the addressees only. If you have received this email in error, please notify the sender. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Johan Henriksson Sent: Monday, November 17, 2008 3:44 AM To: [hidden email] Subject: Re: image analysis-distribution of protein Chris Tully wrote: > Anchal, > > Regardless of which software you are using, you need some way of > isolating the ER and Golgi from the rest of the cell. You may want to > consider fixing the cells and using a membrane stain for isolate the > ER and Golgi based on shape, or you will need to express a third FP in > one of the proteins that populates the ER and Golgi membranes. With a > seperate channel it should be relatively easy to derive ROIs from the > membrane channel for use in measuring the Protein A distribution in > the main image. Now, this technique is only partially accurate. > Since both the ER and Golgi are relatively small bodies that do not > fill the entire vertical space of the cell you really need to look at > this problem in 3D. You will need a lot of slices because you want to > get as close to a cubic voxel as possible. > > I am sure there are some 3D tools for Image J but I would recommend > that you also check out commercial software such as 3D Constructor > (for Image-Pro Plus; http://www.mediacy.com <http://www.mediacy.com>), > Volocity (http://www.improvision.com/products/volocity/), and Imaris > (http://www.bitplane.com/). > or the newer open source packages, Endrov, OME and Bioimagexd /Johan -- -- ------------------------------------------------ Johan Henriksson MSc Engineering PhD student, Karolinska Institutet http://mahogny.areta.org http://www.endrov.net ########################################### This email and any attachment may contain information which is private and confidential and is intended for the addressee only. If you have received this email in error, please destroy it and notify the sender by return email. |
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