Hi All,
I have question about colocalization analysis for 3 channels. I attached an image to make my questions more easily understand. I have 3 images by confocol, from 3 channeal, green, red and blue. I want to measure the colocalization for red and green,(which will be yellow after merge image), however, I want to check the rate between this yellow part/blue. The software by leica can only analysis 2 channels, this really drived my creazy, any help on this would be very appreciated.
Thanks!
Hi Monica,
I have attached a screenshot demoing how MIPAR's Image Processor can create a recipe to measure the area of the blue and the area of yellow at once automatically. I have also attached this recipe file which you may load into the Image Processor and have the analysis automatically performed. I also show a screenshot of the Layers window where you can see the blue and yellow identified areas (overlaid on a grayscale version of the original for clarity).
I am not sure if this is exactly what you meant, but any modifications to this analysis can certainly be made. If you meant you needed the area of the red, green, and blue circles (without or with the red+green area considered in the blue circle) that could be accomplished as well.
Please let me know what, if any, changes need to be made and will be happy to make them.
Cheers,
John
Hi Monica,
An other alternative is CellProfiler, which has multiple modules to measure colocalization depending on your experiment or images. We used it for a large amount of confocal images and the results were quite reliable compared to manual evaluation in AxioVision. You'll find more details and an example pipeline at http://cellprofiler.org/examples.html#Colocalization
Best,
Daniel
I can try to help you using some Matlab code but i need a real image example and not an illustration.
Dear Monica Yiyun Jiang,
Look the links, please.
http://imagej.net/Colocalization_Analysis
http://www.ecu.edu/cs-hs/bsomresearchgradstudies/CoreImagingCenter/upload/Colocalization_AIM_ZEN.pdf
Regards, Shafagat
Hi there, could anyone please tell me the correct setting for colocalization in confocal? Does the pinhole should remain exactly same for all the channels or we just need to adjust pinhole/ detector gain/ offset seperately for different channels which suits best to them and then capture image by having the same focus ( I mean -- not changing the focus manually)?
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