advice on counting objects
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear listers, I was wondering if anybody could give me some advice on how to count the objects in the image as attached. They were cross sections of inner segments of rods and the goal is to get the total number of them. It seemed a easy job at the beginning. But after I started, I realizee that it is tricky. The intensity variation of the objects is big and the boundaries of the cells are not clear. I am not much of an image processing person. I was wondering if anybody could give me some advice on how to proceed. Thank you very much. Xinyu Zhao Biomedical Imaging Core Lab B110 Richards Building School of Medicine University of Pennsylvania 37th and Spruce Street Philadelphia, PA 19104 |
#1978, ODA, 1,000um from ONH, 40X.tif (2M)  
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Glen MacDonald-2 |
Re: advice on counting objects
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Are you using confocal or widefield, was that image a single plane or a brightest point projection? what is the label and mountant? Glen Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] ************************************************************************ ****** The box said "Requires WindowsXP or better", so I bought a Macintosh. ************************************************************************ ****** On Feb 6, 2008, at 12:28 PM, Xinyu Zhao wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > > Dear listers, > > I was wondering if anybody could give me some advice on how to > count the > objects in the image as attached. They were cross sections of > inner segments > of rods and the goal is to get the total number of them. > > It seemed a easy job at the beginning. But after I started, I > realizee that it > is tricky. The intensity variation of the objects is big and the > boundaries of > the cells are not clear. > > I am not much of an image processing person. I was wondering if > anybody could > give me some advice on how to proceed. > > Thank you very much. > > Xinyu Zhao > Biomedical Imaging Core Lab > B110 Richards Building > School of Medicine > University of Pennsylvania > 37th and Spruce Street > Philadelphia, PA 19104<#1978, ODA, 1,000um from ONH, 40X.tif> |
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heckman@bgnet.bgsu.edu |
Re: advice on counting objects
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In reply to this post by Xinyu Zhao-2
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Zhao- The first thing to explore would be the "edge" filter or kernel. If you have any software, such as Image ProPlus, MetaMorph, etc. (don't mean to leave anyone out), it would probably let you execute this function and see if you then have sharp boundaries. There is probably a plug-in for ImageJ as well, which maybe someone can point out where it can be found. (I couldn't really see your image, as my computer treated it as a document.) C. > Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > >Dear listers, > > I was wondering if anybody could give me some advice on how to count the >objects in the image as attached. They were cross sections of inner segments >of rods and the goal is to get the total number of them. > >It seemed a easy job at the beginning. But after I started, I >realizee that it >is tricky. The intensity variation of the objects is big and the >boundaries of >the cells are not clear. > >I am not much of an image processing person. I was wondering if anybody could >give me some advice on how to proceed. > >Thank you very much. > >Xinyu Zhao >Biomedical Imaging Core Lab >B110 Richards Building >School of Medicine >University of Pennsylvania >37th and Spruce Street >Philadelphia, PA 19104 > >Attachment converted: Macintosh HD:#1978, ODA, 1,000um f#90B94.tif >(TEXT/ttxt) (00090B94) -- Carol A. Heckman, Ph.D. Director, Center for Microscopy & Microanalysis and Professor of Biological Sciences Bowling Green State University Bowling Green, OH 43403 USA website: http://www.bgsu.edu/departments/biology/facilities/MnM |
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Deanne Veronica Catmull |
Re: advice on counting objects
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In reply to this post by Xinyu Zhao-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Xinyu, I am no expert myself although I have just been to an imaging workshop where they had a couple of talks which discussed image analysis for counting and I know one in particular used a particular algorithm for discriminating the edges of the cells or particles of interest. You may want to try emailing the speaker Jean-Christophe Olivio-Marin from the Pasteur Institute in Paris. What is your email address, I will pass it on to you? Kind regards, Deanne. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Xinyu Zhao Sent: Thursday, February 07, 2008 7:29 AM To: [hidden email] Subject: advice on counting objects Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear listers, I was wondering if anybody could give me some advice on how to count the objects in the image as attached. They were cross sections of inner segments of rods and the goal is to get the total number of them. It seemed a easy job at the beginning. But after I started, I realizee that it is tricky. The intensity variation of the objects is big and the boundaries of the cells are not clear. I am not much of an image processing person. I was wondering if anybody could give me some advice on how to proceed. Thank you very much. Xinyu Zhao Biomedical Imaging Core Lab B110 Richards Building School of Medicine University of Pennsylvania 37th and Spruce Street Philadelphia, PA 19104 |
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In reply to this post by Xinyu Zhao-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Xinyu,
First, I think you will make your analysis easier and improve your counts by (a) using a confocal (or deconvolution), and/or (b) starting with a more even illumination field (by adjusting your scope fluorescence illumination, or taking a reference image and using it to perform background correction. The dynamic range/contrast of the image seems quite low... maybe you could try to improve that too (better lens...? use camera binning... ?) Then, here's a quick and dirty method using imagej ( http://rsb.info.nih.gov/ij/ ): 0. Open your image 1. Process > Subtract background (radius = 50) (flattens background) 2. Filter > Median (radius 2) (smoothes image) 3. Threshold (30-255), Apply (select thresholded pixels foreground color, remaining pixels background color, black foreground/white background) (thresholds image; play around to see which values work best for your image) 4. Process > Binary >Watershed (separates clusters... to some extent) 5. Go to Analyze > Set measurements and define the parameters to measure. If you just want a count, you may uncheck everything. I recommend checking "Area" and "Display Label" Redirect: None 6. Analyze > Analyze particles; Check all boxes, and select Show outlines 7. Copy data to Excel. From the table of results, you can list particles by size, and you may be able to tell which of those are clusters (just measure the average size of a cell and do a ratio). An even quicker and dirtier way is to measure carefully the surface area of a sample of well-separated cells (as shown above; just pick a good region and get the numbers for the cells that are well separated), threshold all the cells, get total positive area (Using Analyse > Measure, or using the summary with the method above), and divide by average cell area to get a count. Not perfect, but it's a start. You can also look for more sophisticated segmentation plugins at the imagej web site. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 = On Feb 6, 2008, at 12:28 PM, Xinyu Zhao wrote:
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In reply to this post by Glen MacDonald-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, Glen, The picture is from one of our users. It was taken somewhere else in the past and the user would like to do the counting in our lab at this stage. I have no knowledge about the lablel and mountant. But I believe widefield was used and it was a single plane image, and the tissue was dog retina, likely whole mount. The objects here are cross-section view of the inner segments of the rod cells. Obviously it was not a good quality picture to start with, but this is all he has got. Thank you very much for your attention. Xinyu Quoting Glen MacDonald <[hidden email]>: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Are you using confocal or widefield, was that image a single plane or > a brightest point projection? what is the label and mountant? > > Glen > Glen MacDonald > Core for Communication Research > Virginia Merrill Bloedel Hearing Research Center > Box 357923 > University of Washington > Seattle, WA 98195-7923 USA > (206) 616-4156 > [hidden email] > > ************************************************************************ > ****** > The box said "Requires WindowsXP or better", so I bought a Macintosh. > ************************************************************************ > ****** > > > On Feb 6, 2008, at 12:28 PM, Xinyu Zhao wrote: > > > Search the CONFOCAL archive at > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > > > > > > Dear listers, > > > > I was wondering if anybody could give me some advice on how to > > count the > > objects in the image as attached. They were cross sections of > > inner segments > > of rods and the goal is to get the total number of them. > > > > It seemed a easy job at the beginning. But after I started, I > > realizee that it > > is tricky. The intensity variation of the objects is big and the > > boundaries of > > the cells are not clear. > > > > I am not much of an image processing person. I was wondering if > > anybody could > > give me some advice on how to proceed. > > > > Thank you very much. > > > > Xinyu Zhao > > Biomedical Imaging Core Lab > > B110 Richards Building > > School of Medicine > > University of Pennsylvania > > 37th and Spruce Street > > Philadelphia, PA 19104<#1978, ODA, 1,000um from ONH, 40X.tif> > > -- |
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Marius Messerli |
Re: advice on counting objects
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In reply to this post by Xinyu Zhao-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal - commercial response - Dear Xinyu, Imaris determined a count of 1859. The procedure: - drop image file into Imaris - create new spot component - step through detection wizzard - open statistics The Imaris Spot detection/analysis component knows how to handle variable image intensity and cell size. Let me know if you are interested in the final image (overlay of your original image with detected spots) or an evaluation copy of Imaris. Regards, Marius Marius MESSERLI, Ph.D. CEO Bitplane http://www.bitplane.com -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Xinyu Zhao Sent: Mittwoch, 6. Februar 2008 21:29 To: [hidden email] Subject: advice on counting objects Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear listers, I was wondering if anybody could give me some advice on how to count the objects in the image as attached. They were cross sections of inner segments of rods and the goal is to get the total number of them. It seemed a easy job at the beginning. But after I started, I realizee that it is tricky. The intensity variation of the objects is big and the boundaries of the cells are not clear. I am not much of an image processing person. I was wondering if anybody could give me some advice on how to proceed. Thank you very much. Xinyu Zhao Biomedical Imaging Core Lab B110 Richards Building School of Medicine University of Pennsylvania 37th and Spruce Street Philadelphia, PA 19104 |
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Nuno Moreno |
Re: advice on counting objects- Course Annoucement
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, I was not intending to make it international but feel free to show up. _______________________________________________________________________ The Cell Imaging and BioInformatics Units are organizing a course about Quantitative image processing. It will be held at the IGC on the bioiformatics computer room in 26 and 27 February. Synopsis: Imaging analysis, though very powerful, its also a subject of several artifacts which have to be taken in account on experimental design and acquisition. Notwithstanding, it is the desirable method for taking information in microscopy, making us focus on the need of this approach, acquisition details and results obtained within this scope. Because it will be "problem oriented", applicants should propose a real or putative problem for the practicals, which will be the most important selection factor. Instructors: Nuno Moreno (Cell Imaging Unit-IGC) Course Organizer José Leal (BioInformatics Unit-IGC) Mónica Dias (Cell Cycle Regulation-IGC) Gabriel Martins (Centro de Biologia Ambiental-FCUL) For applying please visit: http://bioinformatics.igc.gulbenkian.pt/events/39/ This course is restricted to 20 people and its free for IGC/ITQB members, having a symbolic fee of 100 Euros for others. See you there, Nuno Moreno Marius Messerli wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > - commercial response - > > Dear Xinyu, > > Imaris determined a count of 1859. > > The procedure: > - drop image file into Imaris > - create new spot component > - step through detection wizzard > - open statistics > > The Imaris Spot detection/analysis component knows how to handle variable > image intensity and cell size. > > Let me know if you are interested in the final image (overlay of your > original image with detected spots) or an evaluation copy of Imaris. > > Regards, > Marius > > Marius MESSERLI, Ph.D. > CEO Bitplane > http://www.bitplane.com > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On > Behalf Of Xinyu Zhao > Sent: Mittwoch, 6. Februar 2008 21:29 > To: [hidden email] > Subject: advice on counting objects > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > > Dear listers, > > I was wondering if anybody could give me some advice on how to count the > objects in the image as attached. They were cross sections of inner > segments of rods and the goal is to get the total number of them. > > It seemed a easy job at the beginning. But after I started, I realizee that > it is tricky. The intensity variation of the objects is big and the > boundaries of the cells are not clear. > > I am not much of an image processing person. I was wondering if anybody > could give me some advice on how to proceed. > > Thank you very much. > > Xinyu Zhao > Biomedical Imaging Core Lab > B110 Richards Building > School of Medicine > University of Pennsylvania > 37th and Spruce Street > Philadelphia, PA 19104 > -- Nuno Moreno Cell Imaging Unit Instituto Gulbenkian de Ciência http://uic.igc.gulbekian.pt http://www.igc.gulbekian.pt phone +351 214464606 fax +351 214407970 |
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Winnok H. De Vos |
Re: advice on counting objects
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In reply to this post by Julio Vazquez
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi, An alternative simplistic approach that might do relatively well
for rough numbers ,would be something like: (first open your image) //setBatchMode(true); title=getTitle(); run("Pseudo Flat Field", "mean=100 32-bit");rename("Flat"); run("Gaussian Blur...", "sigma=3");rename("Blur"); setAutoThreshold(); run("Find Maxima...", "noise=0 output=[Segmented
Particles] exclude above"); selectWindow("Blur");close(); rename("particles");run("Invert"); run("Analyze Particles...", "size=0-Infinity
circularity=0.00-1.00 show=Outlines display exclude clear record"); selectWindow("particles");close(); selectWindow("Drawing of
particles");rename("outlines"); run("Invert"); run("RGB Merge...", "red=outlines
green=["+title+"] blue=*None* gray=*None*"); rename("output"); //setBatchMode("exit and display"); As you can see it is still is prone to include some errors, due
to smoothing, especially at the edges, because they are blurred (no plan lens?).
You might want to consider clipping of the edges , e.g., by selecting a rectangular
ROI for analysis. Kind regards,Winnok From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Julio Vazquez Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Xinyu, First,
I think you will make your analysis easier and improve your counts by (a) using
a confocal (or deconvolution), and/or (b) starting with a more even
illumination field (by adjusting your scope fluorescence illumination, or
taking a reference image and using it to perform background
correction. The dynamic range/contrast of the image seems quite
low... maybe you could try to improve that too (better lens...? use
camera binning... ?) Then, here's a quick and dirty method using imagej
( http://rsb.info.nih.gov/ij/
): 0. Open your image 1.
Process > Subtract background (radius = 50) (flattens background) 2.
Filter > Median (radius 2) (smoothes image) 3.
Threshold (30-255), Apply (select thresholded pixels foreground color,
remaining pixels background color, black foreground/white background)
(thresholds image; play around to see which values work best for your image) 4.
Process > Binary >Watershed (separates clusters... to some extent) 5.
Go to Analyze > Set measurements and define the parameters to measure. If
you just want a count, you may uncheck everything. I recommend checking
"Area" and "Display Label" Redirect: None 6.
Analyze > Analyze particles; Check all boxes, and select Show outlines 7.
Copy data to Excel. From the table of results, you can list particles by size,
and you may be able to tell which of those are clusters (just measure the
average size of a cell and do a ratio). An even quicker and dirtier way is to measure carefully the
surface area of a sample of well-separated cells (as shown above; just pick a
good region and get the numbers for the cells that are well separated),
threshold all the cells, get total positive area (Using Analyse > Measure,
or using the summary with the method above), and divide by average cell area to
get a count. Not
perfect, but it's a start. You can also look for more sophisticated
segmentation plugins at the imagej web site. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 = On Feb 6, 2008, at 12:28 PM, Xinyu Zhao wrote:
Search the CONFOCAL archive at Dear listers, I was
wondering if anybody could give me some advice on how to count the objects in the image as attached. They were cross sections of inner
segments of rods and the goal is to get the total number of them. It seemed a easy job at the beginning. But after I started, I realizee that
it is tricky. The
intensity variation of the objects is big and the boundaries of the cells are not clear. I am not much of an image processing person. I was wondering if anybody could give me some advice on how to proceed. Thank you very much. Xinyu Zhao Biomedical Imaging Core Lab B110 Richards Building School of Medicine University of Pennsylvania 37th and Spruce Street Philadelphia, PA 19104<#1978, ODA, 1,000um from ONH,
40X.tif> No virus found in this incoming message. No virus found in this outgoing message. |
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David Barnes-2 |
Re: advice on counting objects
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In reply to this post by Xinyu Zhao-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
On Jan. 23rd, there was a webinar on counting and tracking. The invitation was posted to this list. It is archived at
Download: https://mediacy.webex.com/mediacy/lsr.php?AT=dw&SP=MC&rID=22563052&rKey=01F4A7CE6F10ED49 and there will be a repeat on Feb 26th. dave On Feb 6, 2008 3:28 PM, Xinyu Zhao <[hidden email]> wrote: Search the CONFOCAL archive at |
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In reply to this post by Xinyu Zhao-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal **Commercial response - Image-Pro Plus method (but would apply to any other software which has these tools)** This image does indeed have a highly variable background, however this can be overcome using a couple of simple tools in Image-Pro Plus from Media Cybernetics (and other places, but this is my comfort zone :) ), the procedure would be: 1) Convert image class to GS8 (this is an 8-bit palette image); 2) Run a Local Max filter on the image (I used a 21x21x1 kernel as this is a 2D image, single pass); 3) Dilate 7x7 (connects local max regions within objects); 4) Use the Count/Size module to count all independent objects, setting clean borders to ALL (to not risk double counting objects). I get a result on this image of 1892 objects. An overlay of the processed image with the original showing nice correlation between processed counted bits and the original data can be found here: ftp://ftp.mediacy.com/uploaded/Xinyu/CountOverlay.jpg (258Kb thus not attached - if you want it directly please ask offline). There are some potential false positives in background regions, so I could tweak the filter settings a bit, but I prefer to leave that as an exercise for the end user. If you want more information or a trial of the software, please let me know. Cheers, Paul Jantzen Product Manager, Analytical Software Products Media Cybernetics, Inc. |
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In reply to this post by Xinyu Zhao-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I have used Imaris exclusively for the visuaization of our 3D Confocal data sets. I followed your procedure for counting spots on this 2D image and was very impressed at the ease and quality of the spot counting even with the highly variable background. I am definitely going to use Imaris more on our 2D data. Thank you Marius! Michael Weis Electron Microscopy & Digital Imaging Pacific Agri-Food Research Centre Agriculture and Agri-Food Canada 4200 Highway 97 Summerland, BC V0H 1Z0 Telephone: 250-494-6410 Facsimile: 250-494-0755 [hidden email] -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Marius Messerli Sent: Thursday, February 07, 2008 12:46 AM To: [hidden email] Subject: Re: advice on counting objects Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal - commercial response - Dear Xinyu, Imaris determined a count of 1859. The procedure: - drop image file into Imaris - create new spot component - step through detection wizzard - open statistics The Imaris Spot detection/analysis component knows how to handle variable image intensity and cell size. Let me know if you are interested in the final image (overlay of your original image with detected spots) or an evaluation copy of Imaris. Regards, Marius Marius MESSERLI, Ph.D. CEO Bitplane http://www.bitplane.com -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Xinyu Zhao Sent: Mittwoch, 6. Februar 2008 21:29 To: [hidden email] Subject: advice on counting objects Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear listers, I was wondering if anybody could give me some advice on how to count the objects in the image as attached. They were cross sections of inner segments of rods and the goal is to get the total number of them. It seemed a easy job at the beginning. But after I started, I realizee that it is tricky. The intensity variation of the objects is big and the boundaries of the cells are not clear. I am not much of an image processing person. I was wondering if anybody could give me some advice on how to proceed. Thank you very much. Xinyu Zhao Biomedical Imaging Core Lab B110 Richards Building School of Medicine University of Pennsylvania 37th and Spruce Street Philadelphia, PA 19104 |
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JOEL B. SHEFFIELD |
Re: advice on counting objects
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In reply to this post by Xinyu Zhao-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I'll throw one more possibility into the mix. I used the ImageJ FFT bandpass filter, set at a high value of 100, and a lower value of 0 to smooth out the background. --very impressive!. I then set a threshold, and ran a watershed algorithm. --ended up with about 1900 particles. This seems higher than some have reported. I used a cutoff of 10 pixels^2, and that may have included some small specks. Perhaps it is worth comparing. Joel Date sent: Wed, 6 Feb 2008 15:28:52 -0500 Send reply to: Confocal Microscopy List <[hidden email]> From: Xinyu Zhao <[hidden email]> Subject: advice on counting objects To: [hidden email] > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > > Dear listers, > > I was wondering if anybody could give me some advice on how to count the > objects in the image as attached. They were cross sections of inner segments > of rods and the goal is to get the total number of them. > > It seemed a easy job at the beginning. But after I started, I realizee that it > is tricky. The intensity variation of the objects is big and the boundaries of > the cells are not clear. > > I am not much of an image processing person. I was wondering if anybody could > give me some advice on how to proceed. > > Thank you very much. > > Xinyu Zhao > Biomedical Imaging Core Lab > B110 Richards Building > School of Medicine > University of Pennsylvania > 37th and Spruce Street > Philadelphia, PA 19104 > -- 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 |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I've got 1656 with similar method in ImageJ. First used FFT, cutting out appr. 80% of the frequency spectrum, using four quadrants (one from each corner) and a small cut from the very centre, but leaving the local maxima corresponding to the periodic image elements. The inverse FFT was then calculated and brightness and contrast improved and then thresholded. The Particle Counting -> Nucleus Counter from Plugins was then used and seemed to work well, giving 1656. I'll run it through Volocity to separate touching objects tomorrow.
Zoltan
On 2/7/08, Joel Sheffield <[hidden email]> wrote:
Search the CONFOCAL archive at -- -- Zoltan Cseresnyes Facility manager, Imaging Suite Dept. of Zoology University of Cambridge Downing Street, Cambridge CB2 3EJ UK Tel.: (++44) (0)1223 769282 Fax.: (++44) (0)1223 336676 |
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