Hello
I am looking for a software which allows me to annotate tumour regions, count the number of foci (spots) and nuclei.
These are breast carcinoma TMA slides stained immunofluroscently using the Sigma Proximity Ligation assay. The fluorescence channels are Alexa Fluor Red 549 and DAPI.
Appreciate any suggestions :)
Hi Malcolm,
Are the images you would like to count single images, or are they z-stacks ?
If they are a single image ImageJ might be your best bet. A while ago I wrote an ImageJ macro to automatically count foci in nuclei. I don't have the script anymore but the basic concept was similar to the one described here (http://microscopy.duke.edu/HOWTO/countfoci.html) and I think with some playing around you should be able to make a version that works for you.
If your images are stacks 3D counting would be the way to go. I know all the tools for this are available in the Imaris image processing software. If you don't have acces to this you might be able to do it with ImageJ. There is a plugin called 3D ImageJ suite which has some pretty cool 3D analysis tools similar to Imaris (http://imagejdocu.tudor.lu/doku.php?id=plugin:stacks:3d_ij_suite:start)
Cheers,
Pieter
CellProfiler for the kind of image you provided. You can also calculate the red stain to dapi ratio.
you may have to write your own analysis pipeline but it is worth it. Excellent software
http://cellprofiler.org/examples/
Hi Malcolm,
I quickly adjusted one of the projects I have on CellProfiler to analyse your image. the PPT shows you what is going on.
Of course it can be enhanced to pick all foci more carefully. The parameters will need to be adjusted on your computer independently but this is an example how you make a pipeline (the .cpproj file) that will allow you to analyse large volumes of images automatically and you will have data sheets (xlsx file).
Hope that helps.
F
Thanks for all your suggestions!
I have been trialing ImageJ and Cell profiler for my analysis.
Thanks fares for the pipeline file (tweaking it at the moment)
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