overlapping 2 sets of ROIs

[itzpapalotl]

Hi,

I am a PhD student and a beginner with ImageJ. I recently started doing immunofluorescence experiments and I am looking for a way how to analyze the pictures.

I have 2 channels, first one with DAPI stained nuclei and the second with some of the cells stained for a cytoskeletal marker. I need to count the number of nuclei that are only in the cells marked in second channel. So far I figured out how to count the total number of nuclei in DAPI channel and I can make ROIs with stained cells from the second channel. Now I need to make an overlap between those 2 sets of ROIs and then count the resulting ROIs (the nuclei that were present only in marked cells).

I tried to search the forum, but I only found topics about overlapping single ROIs, not whole sets of them.

I would be very grateful for any kind of advice.

Lenka

[gankaku]

Hi Lenka

Potentially you could post a corresponding image from each channel to get
an idea how it looks like in real life. What you can use for an analysis
like this is the binary feature extractor from the BioVoxxel toolbox. You
can either use Fiji and activate the BioVoxxel update site or download the
packge from http://www.biovoxxel.de/macros.html.
Instructions are under
http://fiji.sc/BioVoxxel_Toolbox#Binary_Feature_Extractor

Instead of Rois you need 2 binary images to proceed. You can get those by
trying to figure out a suitable auto threshold for your images. If you post
some, I or the community might be able to guide you better through a
potential procedure.

Regards,
Jan
Am 06.08.2014 11:38 schrieb "itzpapalotl" <[hidden email]>:

> Hi,
>
> I am a PhD student and a beginner with ImageJ. I recently started doing
> immunofluorescence experiments and I am looking for a way how to analyze
> the
> pictures.
>
> I have 2 channels, first one with DAPI stained nuclei and the second with
> some of the cells stained for a cytoskeletal marker. I need to count the
> number of nuclei that are only in the cells marked in second channel. So
> far
> I figured out how to count the total number of nuclei in DAPI channel and I
> can make ROIs with stained cells from the second channel. Now I need to
> make
> an overlap between those 2 sets of ROIs and then count the resulting ROIs
> (the nuclei that were present only in marked cells).
>
> I tried to search the forum, but I only found topics about overlapping
> single ROIs, not whole sets of them.
>
> I would be very grateful for any kind of advice.
>
> Lenka
>
>
>
> --
> View this message in context:
> http://imagej.1557.x6.nabble.com/overlapping-2-sets-of-ROIs-tp5009043.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[itzpapalotl]

Hi Jan,

thank you very much for your help.

I tried the binary feature extractor and it is very close to what I need. After thresholding images and converting them to binary, I would like to perform a selection of the particles based on size and circularity (similar to what the function Analyze particles does).
For example, in DAPI image I would like to exclude small artifacts that would be otherwise counted as particles in the final extracted image.
Also, in the image of stained cells I would like to exclude small round bright spots (dead /non attached cells), that often also have DAPI signal and therefore they stay in the final extracted image and represent false positive.

Here is link for 1 image from each channel:

http://www.sendspace.com/filegroup/Br4Yc7kwPcC6EweG7yZG5Q

Best regards,

Lenka