Analysis of confocal images: Pixel count in color range of entire stacks

[Lucas]

Hello,

I would like analyze my confocal images by counting pixels of a certain color range in each stack. Thereby I want to quantify the total amount of signal (fluorescent cells) in each channel (gfp/cfp/yfp etc.).

So far I manage to open the .lif (Leica SP5 confocal) files using the LOCI Bioformat plugin and to isolate one channel by using Image>Dublicate. Subsequently I am trying to use the Versatile Wand plugin which nicely selects objects of given color range (in my case the fluorescent cells). However, the tool only makes a selection in the actual plane and not for the entire stack (calculated separately for each plane). Is there a way to count the pixels of interest in the entire stack at once? And how can I finally measure the number of pixels in that selection?

I would be very very happy about any advice or alternative strategy. Please consider that I am not an ImageJ expert (so far;))

Thanks!!

Lucas

[Christian Liebig-2]

Hi,

- go to Analyze> Set measurements
- check the "Area" and the "Limit to threshold" box
- go to Image>Adjust>Threshold
- open the "Set" dialogue and set the upper and lower value of your
desired intensity range
- click on the image and mark the whole image with Ctrl-A
- open the ROI manager (in Analyze>Tools)
- press the "Add" button in the ROI Manager and choose MultiMeasure from
the "More" menu

If I got your request right, this should do the job.
Hope this helps, kind regards,
Christian

On 25.06.2013 19:06, Lucas wrote:

> Hello,
>
> I would like analyze my confocal images by counting pixels of a certain
> color range in each stack. Thereby I want to quantify the total amount of
> signal (fluorescent cells) in each channel (gfp/cfp/yfp etc.).
>
> So far I manage to open the .lif (Leica SP5 confocal) files using the LOCI
> Bioformat plugin and to isolate one channel by using Image>Dublicate.
> Subsequently I am trying to use the Versatile Wand plugin which nicely
> selects objects of given color range (in my case the fluorescent cells).
> However, the tool only makes a selection in the actual plane and not for the
> entire stack (calculated separately for each plane). Is there a way to count
> the pixels of interest in the entire stack at once? And how can I finally
> measure the number of pixels in that selection?
>
> I would be very very happy about any advice or alternative strategy. Please
> consider that I am not an ImageJ expert (so far;))
>
> Thanks!!
>
> Lucas
>
>
>
>
> --
> View this message in context: http://imagej.1557.x6.nabble.com/Analysis-of-confocal-images-Pixel-count-in-color-range-of-entire-stacks-tp5003628.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
Christian Liebig, PhD

Light Microscopy Facility
Max-Planck-Institute for Developmental Biology
Spemannstrasse 35
D-72076 Tuebingen
Germany

Phone:  [phone]
e-mail: [hidden email]
e-mail2: [hidden email]

--
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[Lucas]

Hi Christian,

perfect, that is exactly the answer I was looking for. The results look promising;) Additionally I only had to choose the "dark background" option in the threshold menu and it worked..

Thanks and greets from the MPI Marburg!

Lucas