Re: counting cells in confocal images
Posted by Citron, Bruce on Jun 07, 2011; 8:46pm
URL: http://imagej.273.s1.nabble.com/counting-cells-in-confocal-images-tp3684340p3684341.html
Dear Leon van Gurp,
This is very doable in a macro. I just ran through this with confocals that
we wanted to score % apoptotic with DAPI or Hoechst marking all nuclei and
Annexin-Alexa488 or PI marking dying cells. So for us, it was just two
channels. It seems as if you also want some intensity info in addition to
counts. All of this is possible.
My recommendations:
1. First, play around with a couple of images and make sure that you can get
the numbers you want by executing existing tools or plugins. You might want
to try Analyze Particles- that works for us. Also check out the ShapeLogic
plugins- they handle multicolored cells very well also.
2. Then it can all be automated and a great way to start with macros is to
record exactly what you did with the macro recorder and you¹ll end up with
macro language that you can then tweak.
3. Also look at existing macros for ideas and I can send you our recent
macro which should be modifiable for your puproses.
What kind of confocal are you collecting these with?
We use Olympus Fluoview and the latest LOCI plugins enable ImageJ to open
the .oib files directly and they cover the realm of microscopy formats.
In terms of your work flow:
- Split channel RGB
This should be accomplishable by the settings for the LOCI plugin- we have
ours set for standard ImageJ and split RGB
- Set threshold so that histogram is fully maxed out from 0 to 255 for each
Channel
//Auto adjust contrast from 0-255 allowing 0.35% of pixels to saturate
run("Enhance Contrast", "saturated=0.35 normalized");
- 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
This might be accomplished by intensity values you¹ll get during particle
counting
- For all 7 images (3x red, 3x green, 1x blue), set threshold again from 0
to 255
//Set threshold to 128 (may need to have previously set to dark background)
setThreshold(128,255);
You will likely need different threshold values
- Count the number of positive cells in each image. Cells are not
necessarily round, but are normally 10-20 pixels in diameter. I¹m also not
entirely sure how to threshold this. There is low background in the images,
but there¹s 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!
//Count positive cells in range 70-400 and 0.8-1.0 circularity
run("Analyze Particles...", "size=70-400 circularity=0.80-1.00 show=Nothing
display exclude clear summarize");
- Give a total area result of all positive cells
The particle analysis will actually provide all of the areas and
intensities. You¹ll end up with a Summary window which can be pasted into
excel. I also have a macro for excel that takes the alternating rows of
Hoechst followed by PI counts and results in an Excel table that has all of
the data side by side. Let me know what you need.
Today we had fun counting a set of images with 20 different slides, 10
confocal images per slide with each confocal image containing the nuclear
and apoptotic staining channels. You¹ll be able to let it rip, adjust some
parameters and repeat until you get the parameters the way you¹d like.
-Bruce
--
Bruce A. Citron, Ph.D., Bay Pines VA