Intensity analysis of a ROI from 2 channels

[dcjoshi]

Hell everyoneI want to measure the intensity of a region of interest (ROI) from 2 channels.
In the attached image I would like to measure the intensity of DAB in the regions of Neurofilaments (alexa fluor 488 fluorescence). Is there any way to pick a ROI in the merge image and get the intensity of that ROI from both DAB and neurofilament ?


Thanks
Dinesh C Joshi

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

[gankaku]

Hi Dinesh,

The topic of quantifying chemical stainings of histological sections (or cells) comes up here once in a while. Nevertheless, there is are some hints/suggestions to this topic e.g. from Gabriel Landini (http://www.dentistry.bham.ac.uk/landinig/software/cdeconv/cdeconv.html) and others (https://list.nih.gov/cgi-bin/wa.exe?A2=IMAGEJ;f516c16a.0902). This is a problem which is true for several chromogens. So, unless you have information about the relationship between amount of staining product and absorbance and this relationship is not close to a linear one between those two, a reliable quantification cannot be achieved.
Besides this there are a many more parameters which influence the reliability of such measurements also in fluorescence microscopy (e.g. even lighting of the field of view, binding efficiency and stochiometry of probe/antibody, background,.....).

Having said this, there are several ways to achieve a ROI from one image and measure intensities in related images.

1.) You can try a thresholding of the image which should define the ROI using the "Image>Adjust>Autothreshold" plugin or if necessary a (more biased) manual threshold. Therefore, you might need to pre-process a copy of the image you want to define the ROI in e.g. using background subtraction (Process>Subtract Background...; if suitable for your image) and consecutive image filters to work out your structures and/or reduce noise (e.g. >Process>Filters>...). Often, a median filter does a good job because it facilitates the later thresholding by preserving the edges of features and thus their shape better.
After thresholding you get a binary image. Using the "Analyze Particles" function you can read in those ROIs in the ROI Manager by ticking the "Add to Manager" box. Using the ROI Manager you can easily transfer all those ROIs after each other or at once to other images and measure pixel intensities.

2.) A second alternative could be the "Versatile Wand" tool from Benjamin Schmid which enables you to select intensities in the complete image in a non-connected fashion (also might need some pre-processing a copy of the image). Once you get a ROI using this tool you can also store it in the ROI Manager and transfer it to other images for measuring purposes.

I hope the second part helps you to get ROIs defined and transfered to other images in generell, but be aware of all the multiple limitations, assumptions and parameters which can influence those measurements.
This is definitely not a complete information and I am sure others can further add tipps and information.

Kind regards,
Jan

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

[dcjoshi]

Hi JanThank you very much for providing some suggestions. I will try both and see if I can extract some results.
I am not expecting a linear relationship between the two intensities but want to locate the axons in DAB channel using the green neurofilaments. My goal is to monitor DAB intensity in axons.


Best Regards
Dinesh
On 07/25/13, Jan Brocher - BioVoxxel  wrote:
> Hi Dinesh,
>
> The topic of quantifying

 chemical stainings of histological sections (or cells) comes up here once in a while. Nevertheless, there is are some hints/suggestions to this topic e.g. from Gabriel Landini (http://www.dentistry.bham.ac.uk/landinig/software/cdeconv/cdeconv.html) and others (https://list.nih.gov/cgi-bin/wa.exe?A2=IMAGEJ;f516c16a.0902). This is a problem which is true for several chromogens. So, unless you have information about the relationship between amount of staining product and absorbance and this relationship is not close to a linear one between those two, a reliable quantification cannot be achieved.

> Besides this there are a many more parameters which influence the reliability of such measurements also in fluorescence microscopy (e.g. even lighting of the field of view, binding efficiency and stochiometry of probe/antibody, background,.....).
>
> Having said this, there are several ways to achieve a ROI from one image and measure intensities in related images.
>
> 1.) You can try a thresholding of the image which should define the ROI using the "Image>Adjust>Autothreshold" plugin or if necessary a (more biased) manual threshold. Therefore, you might need to pre-process a copy of the image you want to define the ROI in e.g. using background subtraction (Process>Subtract Background...; if suitable for your image) and consecutive image filters to work out your structures and/or reduce noise (e.g. >Process>Filters>...). Often, a median filter does a good job because it facilitates the later thresholding by preserving the edges of features and thus their shape better.
> After thresholding you get a binary image. Using the "Analyze Particles" function you can read in those ROIs in the ROI Manager by ticking the "Add to Manager" box. Using the ROI Manager you can easily transfer all those ROIs after each other or at once to other images and measure pixel intensities.
>
> 2.) A second alternative could be the "Versatile Wand" tool from Benjamin Schmid which enables you to select intensities in the complete image in a non-connected fashion (also might need some pre-processing a copy of the image). Once you get a ROI using this tool you can also store it in the ROI Manager and transfer it to other images for measuring purposes.
>
> I hope the second part helps you to get ROIs defined and transfered to other images in generell, but be aware of all the multiple limitations, assumptions and parameters which can influence those measurements.
> This is definitely not a complete information and I am sure others can further add tipps and information.
>
> Kind regards,
> Jan
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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