Re: counting cells in confocal images

Posted by Nathaniel Ryckman on Jun 06, 2011; 5:21pm
URL: http://imagej.273.s1.nabble.com/counting-cells-in-confocal-images-tp3684340p3684344.html

Here are some menu commands that will help you achieve what you are trying to achieve:

1) Image->Color->Split Channels
2) Image->Adjust->Threshold (set Threshold)
3) Process->Binary->Watershed
4) (Nuclei Counter) http://www.bioimage.ucsb.edu/downloads/automatic-nuclei-counter-plug-in-for-imagej

5) Analyze->Analyze Particle
6) Analyze->Measure


I think the ITCN plugin (nuclei counter) works fairly well for counting cells.

The problem you might run into is measuring the area for the positive cells. In theory, you should be able to follow steps 1-3 and then 5-6 to achieve that goal. I would say try that first, and, if it doesn't work, post a new post.

By the way, are you a programmer? If you have little or no programming experience, I suggest you learn the imageJ macro language. If you have experience programming, I suggest you learn how to create a plugin.

Good luck!

leonliev wrote

Hi all,

Sorry, i already posted this once, but it did not get accepted by the mailinglist due to my own stupidity. one more try:

I’m pretty new to ImageJ, but I guess I already need some professional results out of the program. I hope there’s somebody who can help me with it. I am analyzing images (TIFF RGB, 787x787 pixels) I obtained doing confocal microscopy, resulting in fluorescent slide images in three colors (red, green, blue). In these slides, there are stained cell nuclei, in different gradations of signal strength for each channel. What I need to do is RGB split the channels, then for the blue channel simply count the amount of cells in it, and for the other two channels count the low, medium and high positive cells. I already tried to make a schematic overview of how the workflow is about to take place:

- Split channel RGB
- Set threshold so that histogram is fully maxed out from 0 to 255 for each channel
- Divide red and green image in three sub images (leave blue channel as it is)
   o Image A including all grayscales from 1 to 85
   o Image B including all grayscales from 86 to 190
   o Image C including all grayscales from 191 to 255
- For all 7 images (3x red, 3x green, 1x blue), set threshold again from 0 to 255
- Count the number of positive cells in each image. Cells are not necessarily round, but are normally 10-20 pixels in diameter. I’m also not entirely sure how to threshold this. There is low background in the images, but there’s always some. Cell nuclei are always more positive, and we need to count them all. I know this isnt a very analytical view, but any advise is welcome!
- Give a total area result of all positive cells

I’m not entirely sure if this is the best way to tackle the subject, any opinions are welcome. Is there anybody who can help me write this as a macro? Is everything I wish to do actually do-able with a macro? All the help you can give me is much appreciated, thanks in advance!

Best regards,
Leon van Gurp