Re: Quantifying Stained Retina

Posted by Joel Sheffield on
URL: http://imagej.273.s1.nabble.com/Quantifying-Stained-Retina-tp5000880p5000890.html

Hi Natalia,

There are several issues with this image, to begin with.  I notice that if
I look  up the histogram of the image, I see that you have ignored about
half of the possible range of pixel intensities when you took the picture.
(min red=154, green=71, blue=177) As a result, you are able to use only
about half of the range of densities to draw distinctions between different
intensities.

Then, there are several different types of staining visible.  1.  the stain
in the onl nuclei (it appears) --quite dense, and mostly uniform throughout
the nucleus, although a bit stellate.  2.  Then there are the nuclei of the
inner nuclear layer, which are stained around the periphery, and perhaps
nucleoli, but are less intense overall.  3.  Then, there are some nuclei in
the choroidal tissues.  4.  Then, there is the general apparent background
stain.

I am not sure which of these you are interested in quantitating.

However, just for fun, I took your image, and inverted the colors, to
generate image b (below).  I then clicked on Color Threshold, which
selected the image as you see it in c.  This, then, has selected all of the
nuclei, but limited itself only to the bits of stain in the inl. There is
no selection in the cytoplasmic material.   You could, I suppose, collect
this data, but I would stress that you would have to be careful to specify
which component you are measuring.  You could, perhaps, do this by creating
an ROI around a specific region.

Joel


On Mon, Nov 19, 2012 at 7:14 PM, Rob van 't Hof
<[hidden email]>wrote:

> So did you use another dye as well to give you the pink colours? if so
> maybe try only the blue stain. If your stain is based on the Prussian Blue
> reaction I'd expect to see very distinct blue, not the pink in your image.
> Have you tried a blood smear as a positive control?
> Rob
>
>
> On 19/11/2012 21:28, Natalia Chacon wrote:
>
>> I'm actually trying to measure blue. Its a dye that attaches to the iron
>> in
>> my cell.
>> I know that ImageJ is able to detect quantities of blue in my image, I'm
>> just having issues getting appropriate measurements.
>>
>> Natalia
>>
>> On Mon, Nov 19, 2012 at 3:16 PM, Rob van 't Hof
>> <[hidden email]>**wrote:
>>
>>  Hi,
>>> I can't see much blue in your sample, mostly pink/purple. is the material
>>> you want to measure the intensely stained pink roughly circular object
>>> that
>>> run diagonally (top left to bottom right) along the image?
>>> bye,
>>> rob
>>>
>>>
>>> On 19/11/2012 19:51, Natalia Chacon wrote:
>>>
>>>  On Mon, Nov 19, 2012 at 1:36 PM, Natalia Chacon
>>>> <[hidden email]>****wrote:
>>>>
>>>>   I mean to quantify the amount of blue staining. Now the staining
>>>>
>>>>> correlates to the amount of iron in my sample.
>>>>> Here is an image of my sample.
>>>>>
>>>>>
>>>>> On Mon, Nov 19, 2012 at 11:33 AM, Rob van 't Hof <
>>>>> [hidden email]> wrote:
>>>>>
>>>>>   Hi Natalia,
>>>>>
>>>>>> A very usefull plugin for analysing stained specimens is Gabriel
>>>>>> Landini's colour deconvolution one (http://www.dentistry.bham.ac.**
>>>>>> ****
>>>>>> uk/landinig/software/cdeconv/******cdeconv.html<http://www.**
>>>>>> dentistry.bham.ac.uk/landinig/****software/cdeconv/cdeconv.**html<http://dentistry.bham.ac.uk/landinig/**software/cdeconv/cdeconv.html>
>>>>>> <http://www.dentistry.**bham.ac.uk/landinig/software/**
>>>>>> cdeconv/cdeconv.html<http://www.dentistry.bham.ac.uk/landinig/software/cdeconv/cdeconv.html>
>>>>>> >
>>>>>>
>>>>>>> **).
>>>>>>>
>>>>>> This is a very powerful plugin to separate different stains.
>>>>>>
>>>>>> When you say "quantify" do mean mean measuring area covered with stain
>>>>>> or
>>>>>> stain intensity (a contentious issue, see documentation to plugin).
>>>>>>
>>>>>> You did not add an example image, so i don't know exactly what you
>>>>>> want
>>>>>> to do. If you add an example image, people like me could create a
>>>>>> basic
>>>>>> macro to show you how to do this kind of stuff.
>>>>>>
>>>>>> bye,
>>>>>> Rob
>>>>>>
>>>>>>
>>>>>> On 19/11/2012 17:07, Natalia Chacon wrote:
>>>>>>
>>>>>>   Hello everyone,
>>>>>>
>>>>>>> I'm a new user of the ImageJ program.
>>>>>>> I'm currently working on a project where I have to quantify the
>>>>>>> amount
>>>>>>> of
>>>>>>> blue staining in an image.
>>>>>>>
>>>>>>> I've already found an article that is similar to what I'm doing,
>>>>>>> except
>>>>>>> its
>>>>>>> quantifying a red stain instead of a blue one.
>>>>>>> (Link provided here:
>>>>>>> http://rsbweb.nih.gov/ij/docs/******examples/stained-sections/******<http://rsbweb.nih.gov/ij/docs/****examples/stained-sections/****>
>>>>>>> index.html<http://rsbweb.nih.**gov/ij/docs/**examples/**
>>>>>>> stained-sections/**index.html<http://rsbweb.nih.gov/ij/docs/**examples/stained-sections/**index.html>
>>>>>>> >
>>>>>>> <http://rsbweb.nih.**gov/ij/**docs/examples/stained-**
>>>>>>> sections/index.html<http://**rsbweb.nih.gov/ij/docs/**
>>>>>>> examples/stained-sections/**index.html<http://rsbweb.nih.gov/ij/docs/examples/stained-sections/index.html>
>>>>>>> >
>>>>>>> )
>>>>>>>
>>>>>>> While this process will work from me, it seems I can't
>>>>>>> *Adjust>Threshold* so
>>>>>>>
>>>>>>> that I can quantify the blue!
>>>>>>>
>>>>>>> Please Advise.
>>>>>>> Thanks,
>>>>>>> Natalia
>>>>>>>
>>>>>>> --
>>>>>>> ImageJ mailing list: http://imagej.nih.gov/ij/list.******html<http://imagej.nih.gov/ij/list.****html>
>>>>>>> <http://imagej.nih.**gov/ij/list.**html<http://imagej.nih.gov/ij/list.**html>
>>>>>>> >
>>>>>>> <http://imagej.nih.gov/**ij/**list.html<http://imagej.nih.gov/**ij/list.html>
>>>>>>> <http://imagej.nih.**gov/ij/list.html<http://imagej.nih.gov/ij/list.html>
>>>>>>> >
>>>>>>>
>>>>>>>   --
>>>>>>>
>>>>>> _____________________________
>>>>>> Dr. Rob van 't Hof
>>>>>> Reader
>>>>>>
>>>>>> Centre for Molecular Medicine
>>>>>> MRC IGMM
>>>>>> University of Edinburgh
>>>>>> Western General Hospital
>>>>>> Crewe Road, Edinburgh EH4 2XU
>>>>>> United Kingdom
>>>>>>
>>>>>> Phone: [phone]
>>>>>> email: [hidden email]
>>>>>> _____________________________
>>>>>>
>>>>>> --
>>>>>> ImageJ mailing list: http://imagej.nih.gov/ij/list.******html<http://imagej.nih.gov/ij/list.****html>
>>>>>> <http://imagej.nih.**gov/ij/list.**html<http://imagej.nih.gov/ij/list.**html>
>>>>>> >
>>>>>> <http://imagej.nih.gov/**ij/**list.html<http://imagej.nih.gov/**ij/list.html>
>>>>>> <http://imagej.nih.**gov/ij/list.html<http://imagej.nih.gov/ij/list.html>
>>>>>> >
>>>>>>
>>>>>>    --
>>>>>
>>>> ImageJ mailing list: http://imagej.nih.gov/ij/list.****html<http://imagej.nih.gov/ij/list.**html>
>>>> <http://imagej.nih.gov/**ij/list.html<http://imagej.nih.gov/ij/list.html>
>>>> >
>>>>
>>>>  --
>>> _____________________________
>>> Dr. Rob van 't Hof
>>> Reader
>>>
>>> Centre for Molecular Medicine
>>> MRC IGMM
>>> University of Edinburgh
>>> Western General Hospital
>>> Crewe Road, Edinburgh EH4 2XU
>>> United Kingdom
>>>
>>> Phone: [phone]
>>> email: [hidden email]
>>> _____________________________
>>>
>>> --
>>> ImageJ mailing list: http://imagej.nih.gov/ij/list.****html<http://imagej.nih.gov/ij/list.**html>
>>> <http://imagej.nih.gov/**ij/list.html<http://imagej.nih.gov/ij/list.html>
>>> >
>>>
>>>  --
>> ImageJ mailing list: http://imagej.nih.gov/ij/list.**html<http://imagej.nih.gov/ij/list.html>
>>
>>
> --
> _____________________________
> Dr. Rob van 't Hof
> Reader
>
> Centre for Molecular Medicine
> MRC IGMM
> University of Edinburgh
> Western General Hospital
> Crewe Road, Edinburgh EH4 2XU
> United Kingdom
>
> Phone: [phone]
> email: [hidden email]
> _____________________________
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.**html<http://imagej.nih.gov/ij/list.html>
>

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs

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