imageJ - quantifying staining intensities

[Babcock, Lyle W]

Hey everyone,

I am new to imageJ, and have been told by my advisor to use imageJ to quantify staining (via immunohistochemistry) intensities in muscle fiber nuclei. I will eventually be analyzing 1000s, potentially 10s of 1000s of nuclei, so I need a more efficient way to do this other than quantifying each nuclei individually via the ROI manager. I followed to power point tutorial on the imageJ website and this is what it is telling me and what is happening:

I split the channels into red, green and blue (I only need the green channel).

Then I converted the image to a threshold image.

Then clicked analyze -> analyze particles

What I have been getting so far is either nothing is counted, or everything i want is counted except it lists everything as an intensity of 255 (for both max and min), with no variation, which I know is not the case in my image.

The only thing that seems to work is instead of going to analyze particles, i go to analyze -> tools -> ROI manager, and click each nuclei individually. Even then, sometimes it measures everything as 0 (which again I know is not the case in my image), or it works just fine, and I have no idea what I'm doing to cause it to go either way.


All I need is a reliable procedure to help me quantify the staining intensities of all the nuclei in my image. Any help will be greatly appreciated!!

Thanks!

Lyle Babcock

Doctoral Research Fellow
Health and Human Performance
Laboratory of Integrated Physiology
University of Houston
919-349-8683

[Lai Ding]

Hello Lyle,

"What I have been getting so far is either nothing is counted, or everything i want is counted except it lists everything as an intensity of 255 (for both max and min), with no variation, which I know is not the case in my image."

 You were measuring the masked image rather than the raw image.  Make a copy of the green channel at the beginning. After the "particle analysis", apply the ROIs to the copied green channel. It should give you the right max/min/variations.

 Best
Lai

Lai Ding, PhD | Optical Imaging Manager
Harvard NeuroDiscovery Center
Harvard Medical School
[phone]
http://www.neurodiscovery.harvard.edu/ 

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Babcock, Lyle W
Sent: Monday, April 09, 2012 12:42 PM
To: [hidden email]
Subject: imageJ - quantifying staining intensities

Hey everyone,

I am new to imageJ, and have been told by my advisor to use imageJ to quantify staining (via immunohistochemistry) intensities in muscle fiber nuclei. I will eventually be analyzing 1000s, potentially 10s of 1000s of nuclei, so I need a more efficient way to do this other than quantifying each nuclei individually via the ROI manager. I followed to power point tutorial on the imageJ website and this is what it is telling me and what is happening:

I split the channels into red, green and blue (I only need the green channel).

Then I converted the image to a threshold image.

Then clicked analyze -> analyze particles

What I have been getting so far is either nothing is counted, or everything i want is counted except it lists everything as an intensity of 255 (for both max and min), with no variation, which I know is not the case in my image.

The only thing that seems to work is instead of going to analyze particles, i go to analyze -> tools -> ROI manager, and click each nuclei individually. Even then, sometimes it measures everything as 0 (which again I know is not the case in my image), or it works just fine, and I have no idea what I'm doing to cause it to go either way.


All I need is a reliable procedure to help me quantify the staining intensities of all the nuclei in my image. Any help will be greatly appreciated!!

Thanks!

Lyle Babcock

Doctoral Research Fellow
Health and Human Performance
Laboratory of Integrated Physiology
University of Houston
919-349-8683

[Joel Sheffield]

Another approach would be to set the threshold on the the image, but don't
convert to binary.  i.e. just leave the threshold settings as you set
them.  Then, analyze particles will pick up all of the densities and
sizes.  We have used this as an exercise to look at variations in dna
content in a growing cell culture.  It is extremely sensitive to uniformity
of background, though.

Joel


On Mon, Apr 9, 2012 at 1:02 PM, Ding, Lai <[hidden email]> wrote:

> Hello Lyle,
>
> "What I have been getting so far is either nothing is counted, or
> everything i want is counted except it lists everything as an intensity of
> 255 (for both max and min), with no variation, which I know is not the case
> in my image."
>
>  You were measuring the masked image rather than the raw image.  Make a
> copy of the green channel at the beginning. After the "particle analysis",
> apply the ROIs to the copied green channel. It should give you the right
> max/min/variations.
>
>  Best
> Lai
>
> Lai Ding, PhD | Optical Imaging Manager
> Harvard NeuroDiscovery Center
> Harvard Medical School
> [phone]
> http://www.neurodiscovery.harvard.edu/
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Babcock, Lyle W
> Sent: Monday, April 09, 2012 12:42 PM
> To: [hidden email]
> Subject: imageJ - quantifying staining intensities
>
> Hey everyone,
>
> I am new to imageJ, and have been told by my advisor to use imageJ to
> quantify staining (via immunohistochemistry) intensities in muscle fiber
> nuclei. I will eventually be analyzing 1000s, potentially 10s of 1000s of
> nuclei, so I need a more efficient way to do this other than quantifying
> each nuclei individually via the ROI manager. I followed to power point
> tutorial on the imageJ website and this is what it is telling me and what
> is happening:
>
> I split the channels into red, green and blue (I only need the green
> channel).
>
> Then I converted the image to a threshold image.
>
> Then clicked analyze -> analyze particles
>
> What I have been getting so far is either nothing is counted, or
> everything i want is counted except it lists everything as an intensity of
> 255 (for both max and min), with no variation, which I know is not the case
> in my image.
>
> The only thing that seems to work is instead of going to analyze
> particles, i go to analyze -> tools -> ROI manager, and click each nuclei
> individually. Even then, sometimes it measures everything as 0 (which again
> I know is not the case in my image), or it works just fine, and I have no
> idea what I'm doing to cause it to go either way.
>
>
> All I need is a reliable procedure to help me quantify the staining
> intensities of all the nuclei in my image. Any help will be greatly
> appreciated!!
>
> Thanks!
>
> Lyle Babcock
>
> Doctoral Research Fellow
> Health and Human Performance
> Laboratory of Integrated Physiology
> University of Houston
> 919-349-8683
>

--


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

[Babcock, Lyle W]

Joel,

Thanks so much for the advice. The particle analyzer is now giving me appropriate max/min variations. However, it is only showing a couple measurements, and there are probably a 100 or so nuclei in the picture. Is there a specific setting that can correct this?

Just to make it easy to understand what I am doing with it, these are the specific commands that I am giving:

Image > color > split channels
(use only the green channel)

Image > adjust > threshold
(i don't click on anything else, it makes the image look completely black with white spots where the nuclei are)

Analyze > analyze particles
(again, i don't click on anything specific, I keep all the default setting)

What comes out is 3 measurements, one of which I am pretty sure is the entire image itself and not an individual nuclei. There are probably 100 or so nuclei I am trying to quantify, so how to I make it do this?

Thanks again!

Lyle Babcock

Doctoral Research Fellow
Health and Human Performance
Laboratory of Integrated Physiology
University of Houston
919-349-8683
________________________________________
From: ImageJ Interest Group [[hidden email]] On Behalf Of Joel B. Sheffield [[hidden email]]
Sent: Monday, April 09, 2012 12:12 PM
To: [hidden email]
Subject: Re: imageJ - quantifying staining intensities

Another approach would be to set the threshold on the the image, but don't
convert to binary.  i.e. just leave the threshold settings as you set
them.  Then, analyze particles will pick up all of the densities and
sizes.  We have used this as an exercise to look at variations in dna
content in a growing cell culture.  It is extremely sensitive to uniformity
of background, though.

Joel


On Mon, Apr 9, 2012 at 1:02 PM, Ding, Lai <[hidden email]> wrote:

> Hello Lyle,
>
> "What I have been getting so far is either nothing is counted, or
> everything i want is counted except it lists everything as an intensity of
> 255 (for both max and min), with no variation, which I know is not the case
> in my image."
>
>  You were measuring the masked image rather than the raw image.  Make a
> copy of the green channel at the beginning. After the "particle analysis",
> apply the ROIs to the copied green channel. It should give you the right
> max/min/variations.
>
>  Best
> Lai
>
> Lai Ding, PhD | Optical Imaging Manager
> Harvard NeuroDiscovery Center
> Harvard Medical School
> [phone]
> http://www.neurodiscovery.harvard.edu/
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Babcock, Lyle W
> Sent: Monday, April 09, 2012 12:42 PM
> To: [hidden email]
> Subject: imageJ - quantifying staining intensities
>
> Hey everyone,
>
> I am new to imageJ, and have been told by my advisor to use imageJ to
> quantify staining (via immunohistochemistry) intensities in muscle fiber
> nuclei. I will eventually be analyzing 1000s, potentially 10s of 1000s of
> nuclei, so I need a more efficient way to do this other than quantifying
> each nuclei individually via the ROI manager. I followed to power point
> tutorial on the imageJ website and this is what it is telling me and what
> is happening:
>
> I split the channels into red, green and blue (I only need the green
> channel).
>
> Then I converted the image to a threshold image.
>
> Then clicked analyze -> analyze particles
>
> What I have been getting so far is either nothing is counted, or
> everything i want is counted except it lists everything as an intensity of
> 255 (for both max and min), with no variation, which I know is not the case
> in my image.
>
> The only thing that seems to work is instead of going to analyze
> particles, i go to analyze -> tools -> ROI manager, and click each nuclei
> individually. Even then, sometimes it measures everything as 0 (which again
> I know is not the case in my image), or it works just fine, and I have no
> idea what I'm doing to cause it to go either way.
>
>
> All I need is a reliable procedure to help me quantify the staining
> intensities of all the nuclei in my image. Any help will be greatly
> appreciated!!
>
> Thanks!
>
> Lyle Babcock
>
> Doctoral Research Fellow
> Health and Human Performance
> Laboratory of Integrated Physiology
> University of Houston
> 919-349-8683
>

--


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

[Gabriel Landini]

On Monday 09 Apr 2012 19:17:48 Babcock, Lyle W wrote:
> What comes out is 3 measurements, one of which I am pretty sure is the
> entire image itself and not an individual nuclei. There are probably 100 or
> so nuclei I am trying to quantify, so how to I make it do this?

Hi,
Be aware that unfortunately immunostains are not stoichiometric, so intensity
measurements will not be very useful/meaningful.

Better to know that now than after spending a lot of time trying to quantify
immunostain intensity.

It only makes sense measuring intensities (to estimate quantity of reactants)
when dealing with stoichiometric stains (like Feulgen for DNA and phalloidin
for actin). Even in those cases you need to calibrate your camera and images
accurately and have controls as well.

Regards

Gabriel

[Joel Sheffield]

It would help if you could post an image that we could look at.

On Mon, Apr 9, 2012 at 2:17 PM, Babcock, Lyle W <[hidden email]>wrote:

> Joel,
>
> Thanks so much for the advice. The particle analyzer is now giving me
> appropriate max/min variations. However, it is only showing a couple
> measurements, and there are probably a 100 or so nuclei in the picture. Is
> there a specific setting that can correct this?
>
> Just to make it easy to understand what I am doing with it, these are the
> specific commands that I am giving:
>
> Image > color > split channels
> (use only the green channel)
>
> Image > adjust > threshold
> (i don't click on anything else, it makes the image look completely black
> with white spots where the nuclei are)
>
> Analyze > analyze particles
> (again, i don't click on anything specific, I keep all the default setting)
>
> What comes out is 3 measurements, one of which I am pretty sure is the
> entire image itself and not an individual nuclei. There are probably 100 or
> so nuclei I am trying to quantify, so how to I make it do this?
>
> Thanks again!
>
> Lyle Babcock
>
> Doctoral Research Fellow
> Health and Human Performance
> Laboratory of Integrated Physiology
> University of Houston
> 919-349-8683
> ________________________________________
> From: ImageJ Interest Group [[hidden email]] On Behalf Of Joel B.
> Sheffield [[hidden email]]
> Sent: Monday, April 09, 2012 12:12 PM
> To: [hidden email]
> Subject: Re: imageJ - quantifying staining intensities
>
> Another approach would be to set the threshold on the the image, but don't
> convert to binary.  i.e. just leave the threshold settings as you set
> them.  Then, analyze particles will pick up all of the densities and
> sizes.  We have used this as an exercise to look at variations in dna
> content in a growing cell culture.  It is extremely sensitive to uniformity
> of background, though.
>
> Joel
>
>
> On Mon, Apr 9, 2012 at 1:02 PM, Ding, Lai <[hidden email]>
> wrote:
>
> > Hello Lyle,
> >
> > "What I have been getting so far is either nothing is counted, or
> > everything i want is counted except it lists everything as an intensity
> of
> > 255 (for both max and min), with no variation, which I know is not the
> case
> > in my image."
> >
> >  You were measuring the masked image rather than the raw image.  Make a
> > copy of the green channel at the beginning. After the "particle
> analysis",
> > apply the ROIs to the copied green channel. It should give you the right
> > max/min/variations.
> >
> >  Best
> > Lai
> >
> > Lai Ding, PhD | Optical Imaging Manager
> > Harvard NeuroDiscovery Center
> > Harvard Medical School
> > [phone]
> > http://www.neurodiscovery.harvard.edu/
> >
> > -----Original Message-----
> > From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> > Babcock, Lyle W
> > Sent: Monday, April 09, 2012 12:42 PM
> > To: [hidden email]
> > Subject: imageJ - quantifying staining intensities
> >
> > Hey everyone,
> >
> > I am new to imageJ, and have been told by my advisor to use imageJ to
> > quantify staining (via immunohistochemistry) intensities in muscle fiber
> > nuclei. I will eventually be analyzing 1000s, potentially 10s of 1000s of
> > nuclei, so I need a more efficient way to do this other than quantifying
> > each nuclei individually via the ROI manager. I followed to power point
> > tutorial on the imageJ website and this is what it is telling me and what
> > is happening:
> >
> > I split the channels into red, green and blue (I only need the green
> > channel).
> >
> > Then I converted the image to a threshold image.
> >
> > Then clicked analyze -> analyze particles
> >
> > What I have been getting so far is either nothing is counted, or
> > everything i want is counted except it lists everything as an intensity
> of
> > 255 (for both max and min), with no variation, which I know is not the
> case
> > in my image.
> >
> > The only thing that seems to work is instead of going to analyze
> > particles, i go to analyze -> tools -> ROI manager, and click each nuclei
> > individually. Even then, sometimes it measures everything as 0 (which
> again
> > I know is not the case in my image), or it works just fine, and I have no
> > idea what I'm doing to cause it to go either way.
> >
> >
> > All I need is a reliable procedure to help me quantify the staining
> > intensities of all the nuclei in my image. Any help will be greatly
> > appreciated!!
> >
> > Thanks!
> >
> > Lyle Babcock
> >
> > Doctoral Research Fellow
> > Health and Human Performance
> > Laboratory of Integrated Physiology
> > University of Houston
> > 919-349-8683
> >
>
>
>
> --
>
>
> 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
>

--


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

[Anda Cornea]

I think your threshold is selecting the background rather than the nuclei.  Make sure you select dark background if you use fluorescence.

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Babcock, Lyle W
Sent: Monday, April 09, 2012 11:18 AM
To: [hidden email]
Subject: Re: imageJ - quantifying staining intensities

Joel,

Thanks so much for the advice. The particle analyzer is now giving me appropriate max/min variations. However, it is only showing a couple measurements, and there are probably a 100 or so nuclei in the picture. Is there a specific setting that can correct this?

Just to make it easy to understand what I am doing with it, these are the specific commands that I am giving:

Image > color > split channels
(use only the green channel)

Image > adjust > threshold
(i don't click on anything else, it makes the image look completely black with white spots where the nuclei are)

Analyze > analyze particles
(again, i don't click on anything specific, I keep all the default setting)

What comes out is 3 measurements, one of which I am pretty sure is the entire image itself and not an individual nuclei. There are probably 100 or so nuclei I am trying to quantify, so how to I make it do this?

Thanks again!

Lyle Babcock

Doctoral Research Fellow
Health and Human Performance
Laboratory of Integrated Physiology
University of Houston
919-349-8683
________________________________________
From: ImageJ Interest Group [[hidden email]] On Behalf Of Joel B. Sheffield [[hidden email]]
Sent: Monday, April 09, 2012 12:12 PM
To: [hidden email]
Subject: Re: imageJ - quantifying staining intensities

Another approach would be to set the threshold on the the image, but don't convert to binary.  i.e. just leave the threshold settings as you set them.  Then, analyze particles will pick up all of the densities and sizes.  We have used this as an exercise to look at variations in dna content in a growing cell culture.  It is extremely sensitive to uniformity of background, though.

Joel


On Mon, Apr 9, 2012 at 1:02 PM, Ding, Lai <[hidden email]> wrote:

> Hello Lyle,
>
> "What I have been getting so far is either nothing is counted, or
> everything i want is counted except it lists everything as an
> intensity of
> 255 (for both max and min), with no variation, which I know is not the
> case in my image."
>
>  You were measuring the masked image rather than the raw image.  Make
> a copy of the green channel at the beginning. After the "particle
> analysis", apply the ROIs to the copied green channel. It should give
> you the right max/min/variations.
>
>  Best
> Lai
>
> Lai Ding, PhD | Optical Imaging Manager Harvard NeuroDiscovery Center
> Harvard Medical School
> [phone]
> http://www.neurodiscovery.harvard.edu/
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of
> Babcock, Lyle W
> Sent: Monday, April 09, 2012 12:42 PM
> To: [hidden email]
> Subject: imageJ - quantifying staining intensities
>
> Hey everyone,
>
> I am new to imageJ, and have been told by my advisor to use imageJ to
> quantify staining (via immunohistochemistry) intensities in muscle
> fiber nuclei. I will eventually be analyzing 1000s, potentially 10s of
> 1000s of nuclei, so I need a more efficient way to do this other than
> quantifying each nuclei individually via the ROI manager. I followed
> to power point tutorial on the imageJ website and this is what it is
> telling me and what is happening:
>
> I split the channels into red, green and blue (I only need the green
> channel).
>
> Then I converted the image to a threshold image.
>
> Then clicked analyze -> analyze particles
>
> What I have been getting so far is either nothing is counted, or
> everything i want is counted except it lists everything as an
> intensity of
> 255 (for both max and min), with no variation, which I know is not the
> case in my image.
>
> The only thing that seems to work is instead of going to analyze
> particles, i go to analyze -> tools -> ROI manager, and click each
> nuclei individually. Even then, sometimes it measures everything as 0
> (which again I know is not the case in my image), or it works just
> fine, and I have no idea what I'm doing to cause it to go either way.
>
>
> All I need is a reliable procedure to help me quantify the staining
> intensities of all the nuclei in my image. Any help will be greatly
> appreciated!!
>
> Thanks!
>
> Lyle Babcock
>
> Doctoral Research Fellow
> Health and Human Performance
> Laboratory of Integrated Physiology
> University of Houston
> 919-349-8683
>

--


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