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Hello everyone,
I have had much success in counting Hoechst-stained nuclei in images of various types of cultured cells in the past using the ITCN plugin, but I am having much difficulty with a current set of images. The difficulty seems to be that there are two populations present, one set is small and brightly stained, and the other is large and more faint. Here is a link to a sample image: https://www.slashtmp.iu.edu/public/download.php?FILE=jasabail/42663HrCy7o I have been unable to get ITCN to reliably count the large nuclei with any settings I've tried, or with masking the small bright nuclei. I've tried a few other things, such as watershed (both the built-in process and a plugin written by Daniel Sage I found searching the archives), but these have not worked. Can anyone suggest a better strategy? Thanks Jason |
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Hi,
On Mar 26, 2010, at 5:17 PM, Jason Bailey wrote: > Hello everyone, > > I have had much success in counting Hoechst-stained nuclei in images > of > various types of cultured cells in the past using the ITCN plugin, > but I am > having much difficulty with a current set of images. The difficulty > seems to > be that there are two populations present, one set is small and > brightly > stained, and the other is large and more faint. Here is a link to a > sample > image: > > https://www.slashtmp.iu.edu/public/download.php?FILE=jasabail/42663HrCy7o > > I have been unable to get ITCN to reliably count the large nuclei > with any > settings I've tried, or with masking the small bright nuclei. I've > tried a > few other things, such as watershed (both the built-in process and a > plugin > written by Daniel Sage I found searching the archives), but these > have not > worked. > ... [show rest of quote] If you only want the brighter population you might try using the RATS segmentation plugin followed by a watershed. We have used it quite a bit with similar images. Unfortunately RATS doesn't handle multiple thresholds so if you want the fainter objects I am not sure what to suggest. You can find it here... http://rsb.info.nih.gov/ij/plugins/rats/index.html Cheers, Ben |
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In reply to this post by Jason Bailey-2
On Friday 26 March 2010, you wrote:
> I have had much success in counting Hoechst-stained nuclei in images of > various types of cultured cells in the past using the ITCN plugin, but I am > having much difficulty with a current set of images. The difficulty seems > to be that there are two populations present, one set is small and > brightly stained, and the other is large and more faint. Here is a link to > a sample image: > > https://www.slashtmp.iu.edu/public/download.php?FILE=jasabail/42663HrCy7o Have a look at the CLAHE plugin http://rsb.info.nih.gov/ij/plugins/clahe/index.html That brings up the dark nuclei and might make it easier to segment them. G. |
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In reply to this post by Jason Bailey-2
Thanks, all!
The RATS plugin did very well at pulling up the bright nuclei, but it cut out the big, darker ones that I was having trouble counting. It will actually work very nicely for another difficult set of images I need to quantify. Using the CLAHE plugin to pre-process images before running ITCN improved the counts quite a bit. I still need to play with the settings a little, but that's a big step in the right direction. And as a bonus, it is fun to watch. Thanks again! Jason |
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