Quantifying Stained Retina

[NatyC]

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/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

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

[Rob van 't Hof-2]

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). 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/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
>

... [show rest of quote]

--
_____________________________
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

[NatyC]

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>).
>> 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/**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>
>>>
>>>
>> --
>> _____________________________
>> 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>
>>
>
>

... [show rest of quote]

... [show rest of quote]

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[Rob van 't Hof-2]

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>).
>>> 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/**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>
>>>>
>>>>
>>> --
>>> _____________________________
>>> 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>
>>>
>>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

... [show rest of quote]

--
_____________________________
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

[carlo bianco]

Hello Natalia,
The deconvolution made by prof Landini is excellent, if you work with well known
stains (histo/ihc) if you must face and manage
 other colors, I suggest you the segmentation plugin,


http://bigwww.epfl.ch/sage/soft/colorsegmentation/
best regards
carlo




________________________________
 Da: Rob van 't Hof <[hidden email]>
A: [hidden email]
Inviato: Lunedì 19 Novembre 2012 18:33
Oggetto: Re: Quantifying Stained Retina
 
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). 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/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
>

... [show rest of quote]

-- _____________________________
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

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

[NatyC]

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://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/****
>>>>> 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>
>>>>> >
>>>>>
>>>>>
>>>>>  --
>>>> _____________________________
>>>> 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>
>

... [show rest of quote]

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

[Rob van 't Hof-2]

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

... [show rest of quote]

--
_____________________________
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

[Joel Sheffield]

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>
>

... [show rest of quote]

... [show rest of quote]

--


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

[NatyC]

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

... [show rest of quote]

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

[Gabriel Landini]

On Tuesday 20 Nov 2012 14:55:39 Natalia Chacon wrote:
> 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.

This is what Prussian, Pearls or Berlin blue looks like:

http://www.jichi.ac.jp/pathology/swfu/d/fe-10.jpeg

Your image looks like all contrast stain to me. So you might not have any iron
compounds there or it is so faint that the contrast stain prevails.

With Berlin blue, you can *demonstrate* that some iron compound is there, but
I am not sure if it can be used to quantify their quantity. Here it says it
cannot, so I would be careful how to interpret the intensity:

http://stainsfile.info/StainsFile/stain/pigment/perls.htm

Regards

Gabriel

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

[Joel Sheffield]

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

... [show rest of quote]

--


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

[NatyC]

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]
>> >>>>>>> _____________________________
>> >>>>>>>
>> >>>>>>> --
>> >>>>>>> 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
>> >
>> > --
>> > 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
>

... [show rest of quote]

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[NatyC]

On 11/28/12, Natalia Chacon <[hidden email]> wrote:

> Dear Mr. Gabriel,
>
> I've enclosed another image that seems to contain more iron in the sample.
> Also, is it still possible to use the Color Deconvolution to get the
> data my mentor desires?
>
> Thanks,
> Natalia
>
> On 11/20/12, Gabriel Landini <[hidden email]> wrote:
>> On Tuesday 20 Nov 2012 14:55:39 Natalia Chacon wrote:
>>> 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.
>>
>> This is what Prussian, Pearls or Berlin blue looks like:
>>
>> http://www.jichi.ac.jp/pathology/swfu/d/fe-10.jpeg
>>
>> Your image looks like all contrast stain to me. So you might not have any
>> iron
>> compounds there or it is so faint that the contrast stain prevails.
>>
>> With Berlin blue, you can *demonstrate* that some iron compound is there,
>> but
>> I am not sure if it can be used to quantify their quantity. Here it says
>> it
>>
>> cannot, so I would be careful how to interpret the intensity:
>>
>> http://stainsfile.info/StainsFile/stain/pigment/perls.htm
>>
>> Regards
>>
>> Gabriel
>>
>> --
>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>>
>

... [show rest of quote]

... [show rest of quote]

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[karo03]

Hi,
this is an interesting thread with many very differing layers of understanding of images!

What about measuring the "amount" of blue, which is IMHU the total extinction of the blue channel, in a selected region, e.g. a layer or a part of it? For standardization the area of the region should be measured too, or if the nuclei are of interest, better the area of the nuclei inside the selected region.

... and so on, step by step a real measurement scheme will be established.

For reliable measurements lots of questions will arise! If "the iron" stained by "Pearls Blue" is in any sense stoichiometric perhaps images should be sampled as pure intensity image using the appropriate filter!

However, I would recommend to measure, as the mentor ask for, and look at the results.
Also mentors are still learning!

Regards
Karsten

Am 28.11.2012 um 21:55 schrieb Natalia Chacon:

> On 11/28/12, Natalia Chacon <[hidden email]> wrote:
>> Dear Mr. Gabriel,
>>
>> I've enclosed another image that seems to contain more iron in the sample.
>> Also, is it still possible to use the Color Deconvolution to get the
>> data my mentor desires?
>>
>> Thanks,
>> Natalia
>>
>> On 11/20/12, Gabriel Landini <[hidden email]> wrote:
>>> On Tuesday 20 Nov 2012 14:55:39 Natalia Chacon wrote:
>>>> 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.
>>>
>>> This is what Prussian, Pearls or Berlin blue looks like:
>>>
>>> http://www.jichi.ac.jp/pathology/swfu/d/fe-10.jpeg
>>>
>>> Your image looks like all contrast stain to me. So you might not have any
>>> iron
>>> compounds there or it is so faint that the contrast stain prevails.
>>>
>>> With Berlin blue, you can *demonstrate* that some iron compound is there,
>>> but
>>> I am not sure if it can be used to quantify their quantity. Here it says
>>> it
>>>
>>> cannot, so I would be careful how to interpret the intensity:
>>>
>>> http://stainsfile.info/StainsFile/stain/pigment/perls.htm
>>>
>>> Regards
>>>
>>> Gabriel
>>>
>>> --
>>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>>>
>>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
> <BB.JPG>

... [show rest of quote]

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[Gabriel Landini]

On Wednesday 28 Nov 2012 20:55:11 Natalia Chacon wrote:
> I've enclosed another image that seems to contain more iron in the sample.
> Also, is it still possible to use the Color Deconvolution to get the
> data my mentor desires?

Hi, I do not think there is a lot of blue in your images, so I doubt that it
can be done reliably.
Look at the colour cube of your image (color inspector 3d) and you will
realise what I mean.

Cheers

Gabriel

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[Joel Sheffield]

Hi All,

First, the stain that Natalie is referring to is known in the histo world
as "Perls Prussian Blue".  A brief description follows:

a stain for ferric iron as in hemosiderins, using potassium ferrocyanide in
acetic acid or dilute hydrochloric acid followed by a red counterstain such
as safranin O or neutral red; various hemosiderins and most mineral irons
give a blue-green reaction, while nuclei stain red.

Note that the procedure often includes a counterstain.  Natalie, do you
know if your procedure includes some other dye, such as those mentioned
above?  If so, that would account for Gabriel's concerns about red in the
image.  Being color blind, I see very little of that color on my monitor,
but there can be lots of variation in monitors.  I will make the
assumption, for the sake of argument, that there actually is a
counterstain, in which case I would recommend that you try the stain
without it.  It might be possible to resolve the contribution of the Perls
stain under those conditions.
One more point, since you are interested in layers, it will be much easier
to quantify the stain per layer if you take your images so that the layers
are horizontal.

Joel

On Wed, Nov 28, 2012 at 5:07 PM, Gabriel Landini <[hidden email]>wrote:

> On Wednesday 28 Nov 2012 20:55:11 Natalia Chacon wrote:
> > I've enclosed another image that seems to contain more iron in the
> sample.
> > Also, is it still possible to use the Color Deconvolution to get the
> > data my mentor desires?
>
> Hi, I do not think there is a lot of blue in your images, so I doubt that
> it
> can be done reliably.
> Look at the colour cube of your image (color inspector 3d) and you will
> realise what I mean.
>
> Cheers
>
> Gabriel
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

... [show rest of quote]

--


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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[NatyC]

Dear Joel,

Unfortunetly we can't go back and restain these images.
The staining was done outside of our lab by a three party it seems.
So that means I'll have to work with what I'm being provided.

Joel you mentioned this might assist me with research.

"One more point, since you are interested in layers, it will be much easier
 to quantify the stain per layer if you take your images so that the layers
 are horizontal."

However, how can I go about doing that?
Do I have to go to the orignal sample and start from there, or is
there a plugin I can use to assist me with this process?

Thanks,
Natalia



On 11/28/12, JOEL B. SHEFFIELD <[hidden email]> wrote:

> Hi All,
>
> First, the stain that Natalie is referring to is known in the histo world
> as "Perls Prussian Blue".  A brief description follows:
>
> a stain for ferric iron as in hemosiderins, using potassium ferrocyanide in
> acetic acid or dilute hydrochloric acid followed by a red counterstain such
> as safranin O or neutral red; various hemosiderins and most mineral irons
> give a blue-green reaction, while nuclei stain red.
>
> Note that the procedure often includes a counterstain.  Natalie, do you
> know if your procedure includes some other dye, such as those mentioned
> above?  If so, that would account for Gabriel's concerns about red in the
> image.  Being color blind, I see very little of that color on my monitor,
> but there can be lots of variation in monitors.  I will make the
> assumption, for the sake of argument, that there actually is a
> counterstain, in which case I would recommend that you try the stain
> without it.  It might be possible to resolve the contribution of the Perls
> stain under those conditions.
> One more point, since you are interested in layers, it will be much easier
> to quantify the stain per layer if you take your images so that the layers
> are horizontal.
>
> Joel
>
> On Wed, Nov 28, 2012 at 5:07 PM, Gabriel Landini
> <[hidden email]>wrote:
>
>> On Wednesday 28 Nov 2012 20:55:11 Natalia Chacon wrote:
>> > I've enclosed another image that seems to contain more iron in the
>> sample.
>> > Also, is it still possible to use the Color Deconvolution to get the
>> > data my mentor desires?
>>
>> Hi, I do not think there is a lot of blue in your images, so I doubt that
>> it
>> can be done reliably.
>> Look at the colour cube of your image (color inspector 3d) and you will
>> realise what I mean.
>>
>> Cheers
>>
>> Gabriel
>>
>> --
>> 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
>

... [show rest of quote]

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[NatyC]

Dear Karsten,

How would you recommend measuring these ROI?

I've tested out the basic Analyze>Set Measurements, Analyze>
Measurement on a Split Channel (RGB). With this process I measured the
area,integrated density, and mean gray density of the Split Channel
Blue image.

The issue with this analysis is that there is no true distiction in
the amount of "Blue"
Rather, all of the values ended up to be the total amount of pixels
that compose the image.

Natalia

On 11/28/12, Karsten <[hidden email]> wrote:

> Hi,
> this is an interesting thread with many very differing layers of
> understanding of images!
>
> What about measuring the "amount" of blue, which is IMHU the total
> extinction of the blue channel, in a selected region, e.g. a layer or a part
> of it? For standardization the area of the region should be measured too, or
> if the nuclei are of interest, better the area of the nuclei inside the
> selected region.
>
> ... and so on, step by step a real measurement scheme will be established.
>
> For reliable measurements lots of questions will arise! If "the iron"
> stained by "Pearls Blue" is in any sense stoichiometric perhaps images
> should be sampled as pure intensity image using the appropriate filter!
>
> However, I would recommend to measure, as the mentor ask for, and look at
> the results.
> Also mentors are still learning!
>
> Regards
> Karsten
>
> Am 28.11.2012 um 21:55 schrieb Natalia Chacon:
>
>> On 11/28/12, Natalia Chacon <[hidden email]> wrote:
>>> Dear Mr. Gabriel,
>>>
>>> I've enclosed another image that seems to contain more iron in the
>>> sample.
>>> Also, is it still possible to use the Color Deconvolution to get the
>>> data my mentor desires?
>>>
>>> Thanks,
>>> Natalia
>>>
>>> On 11/20/12, Gabriel Landini <[hidden email]> wrote:
>>>> On Tuesday 20 Nov 2012 14:55:39 Natalia Chacon wrote:
>>>>> 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.
>>>>
>>>> This is what Prussian, Pearls or Berlin blue looks like:
>>>>
>>>> http://www.jichi.ac.jp/pathology/swfu/d/fe-10.jpeg
>>>>
>>>> Your image looks like all contrast stain to me. So you might not have
>>>> any
>>>> iron
>>>> compounds there or it is so faint that the contrast stain prevails.
>>>>
>>>> With Berlin blue, you can *demonstrate* that some iron compound is
>>>> there,
>>>> but
>>>> I am not sure if it can be used to quantify their quantity. Here it
>>>> says
>>>> it
>>>>
>>>> cannot, so I would be careful how to interpret the intensity:
>>>>
>>>> http://stainsfile.info/StainsFile/stain/pigment/perls.htm
>>>>
>>>> Regards
>>>>
>>>> Gabriel
>>>>
>>>> --
>>>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>>>>
>>>
>>
>> --
>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>> <BB.JPG>
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

... [show rest of quote]

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[karo03]

Ok, the first step you did. Clearly the integrated density reflects the region size.
 
Hence what is the related area to get a reliable estimator of mean density. I would recommend either the cells, if they are segmentable, or the inverse of the white background, e.g. taken from the blue channel. This makes sense for regions, where the cells are recognizable.

The regions of each layer should be defined as large as possible (area=A). To correct a bit the areas, measure also the  white area (background, BG) inside the regions.
Than calculate: mean density=integrated density / (A-BG) per region.

I don't know how ImageJ calculates integrated density, I would recommend to transform the channel image to "extinction" by
run("32-bit");
run("Divide...", "value=255");
# 255 is the white incoming light, possibly it is a bit less.
# The pixel values of your image are the transmitted light.
run("Log");
run("Multiply...", "value=-1");
//run("Brightness/Contrast..."); # to make the image content visible
and measure than the mean gray value. Than is integrated density = mean gray value * (A) and mean density can than be calculated as given above. Following the infos from Joel it makes sense to do these measurements on each channel! Possibly also a color deconvolution (PCA ?) could make sense to discriminate the cell stain from the iron stain.

If this/these "mean density" does/do not differ enough, meaning that the amount of iron per layer is not differentiable with these measures, the next step starts: Up to now it is assumed, that the section thickness is constant and the stain reaches homogeneously the iron in the whole material. This has to be proven, e.g. by testing some samples (not the delivered images) from somebody with expertise in (quantitative) microscopy! Critical is the section thickness!

I think with the numbers calculated as described for a senseful set of images (with layers) per group (?), it will be not completely easy to state "non-discriminability" of iron content!

Best regards
Karsten

Am 29.11.2012 um 21:05 schrieb Natalia Chacon:

> Dear Karsten,
>
> How would you recommend measuring these ROI?
>
> I've tested out the basic Analyze>Set Measurements, Analyze>
> Measurement on a Split Channel (RGB). With this process I measured the
> area,integrated density, and mean gray density of the Split Channel
> Blue image.
>
> The issue with this analysis is that there is no true distiction in
> the amount of "Blue"
> Rather, all of the values ended up to be the total amount of pixels
> that compose the image.
>
> Natalia
>
> On 11/28/12, Karsten <[hidden email]> wrote:
>> Hi,
>> this is an interesting thread with many very differing layers of
>> understanding of images!
>>
>> What about measuring the "amount" of blue, which is IMHU the total
>> extinction of the blue channel, in a selected region, e.g. a layer or a part
>> of it? For standardization the area of the region should be measured too, or
>> if the nuclei are of interest, better the area of the nuclei inside the
>> selected region.
>>
>> ... and so on, step by step a real measurement scheme will be established.
>>
>> For reliable measurements lots of questions will arise! If "the iron"
>> stained by "Pearls Blue" is in any sense stoichiometric perhaps images
>> should be sampled as pure intensity image using the appropriate filter!
>>
>> However, I would recommend to measure, as the mentor ask for, and look at
>> the results.
>> Also mentors are still learning!
>>
>> Regards
>> Karsten
>>
>> Am 28.11.2012 um 21:55 schrieb Natalia Chacon:
>>
>>> On 11/28/12, Natalia Chacon <[hidden email]> wrote:
>>>> Dear Mr. Gabriel,
>>>>
>>>> I've enclosed another image that seems to contain more iron in the
>>>> sample.
>>>> Also, is it still possible to use the Color Deconvolution to get the
>>>> data my mentor desires?
>>>>
>>>> Thanks,
>>>> Natalia
>>>>
>>>> On 11/20/12, Gabriel Landini <[hidden email]> wrote:
>>>>> On Tuesday 20 Nov 2012 14:55:39 Natalia Chacon wrote:
>>>>>> 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.
>>>>>
>>>>> This is what Prussian, Pearls or Berlin blue looks like:
>>>>>
>>>>> http://www.jichi.ac.jp/pathology/swfu/d/fe-10.jpeg
>>>>>
>>>>> Your image looks like all contrast stain to me. So you might not have
>>>>> any
>>>>> iron
>>>>> compounds there or it is so faint that the contrast stain prevails.
>>>>>
>>>>> With Berlin blue, you can *demonstrate* that some iron compound is
>>>>> there,
>>>>> but
>>>>> I am not sure if it can be used to quantify their quantity. Here it
>>>>> says
>>>>> it
>>>>>
>>>>> cannot, so I would be careful how to interpret the intensity:
>>>>>
>>>>> http://stainsfile.info/StainsFile/stain/pigment/perls.htm
>>>>>
>>>>> Regards
>>>>>
>>>>> Gabriel
>>>>>
>>>>> --
>>>>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>>>>>
>>>>
>>>
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>>
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Karsten
[hidden email]

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