Re: Quantifying Stained Retina

Posted by NatyC on
URL: http://imagej.273.s1.nabble.com/Quantifying-Stained-Retina-tp5000880p5000992.html

To Joel,

My mentor wants me to quantify the amount of blue staining within each
cell layer.
My denominator being the individual layers I select in the retina and
then in the whole image.

Natalia

On 11/20/12, JOEL B. SHEFFIELD <[hidden email]> wrote:

> Hi Natalia,
>
> I'm still a little confused about what you want to quantify.  Do you want
> to count the number of positive nuclei?  Do you want to know what the total
> binding of stain is, regardless of structure?
>
> If so, what is your denominator?  Is it the area of the section that is
> retina, or is it the visible area in the image, including non-retinal
> tissue?
>
> On Tue, Nov 20, 2012 at 9:55 AM, Natalia Chacon
> <[hidden email]>wrote:
>
>> Hello Rob and Joel,
>>
>> Rob, my mentor told me she used a stain called "Pearls Blue"
>>
>> Joel, I'm interested in quantifiying most of the stainings visible.
>> (the background staining I don't desire to quantify.)
>>
>> What you're stating though does correlate with what my mentor has
>> informed
>> me.
>> That the onl nuclei would be the most dense.
>> This would be due to the way the protien settles.
>>
>> Thanks for the advice Joel.
>>
>> Natalia
>>
>> On 11/19/12, JOEL B. SHEFFIELD <[hidden email]> wrote:
>> > 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]
>> >>>>>>> _____________________________
>> >>>>>>>
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>> >>>>>>> >
>> >>>>>>> <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>
>> >>>>>>> >
>> >>>>>>>
>> >>>>>>>    --
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>> >>>>> >
>> >>>>>
>> >>>>>  --
>> >>>> _____________________________
>> >>>> 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]
>> >>>> _____________________________
>> >>>>
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>> >>>> >
>> >>>>
>> >>>>  --
>> >>> ImageJ mailing list:
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>> >>>
>> >>>
>> >> --
>> >> _____________________________
>> >> 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:
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>> >
>> >>
>> >
>> >
>> >
>> > --
>> >
>> >
>> > 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
>> >
>> > --
>> > ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>> >
>>
>> --
>> ImageJ mailing list: 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
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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