measuring fluorescent intensity

[nurul]

Hi there,
 
I want to measure the fluorescence of dye in various
part of eye. I'm attaching examples of my pictures. (Tv79, I want to
measure intensity of whole picture, while in TV104, I want to measure
selected structure only)
However, I am very confused using Image J. Should I take the int. Density measurement or mean gray area?
Is
 the measurement is affected by how large the area of cell/tissue? If
so, this means I need to standardize area size in all my sample ( which
is also another problem as I'm not able to do that..)
 
Can anyone
 care to guide me step by step way in measuring intensity for my case?
(I'm currently only using technique as written in this blog : http://sciencetechblog.com/2011/05/24/measuring-cell-fluorescence-using-imagej/ )
 
If anyone can guide me, it is very helpful. Thank you everyone in advance
 
Dr. Nurul Alimah Abdul Nasir
MBBS (Malaya)
Trainee Lecturer, Pharmacology, FOM UiTM
[phone]
[phone]

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

[nurul]

Thanks Charles,
I'm sorry but I'm still quite blur.. I'm not sure about the calibrate function, and those calibration doesn't involve coloured image...
Should I calibrate the image,then?
Can't I just measure the intensity straight away?

Thanks..


Dr. Nurul Alimah Abdul Nasir
MBBS (Malaya)
Trainee Lecturer, Pharmacology, FOM UiTM
[phone]
[phone]




________________________________
 From: "Anderson, Charles (DNR)" <[hidden email]>
To: Nurul Alimah Abdul Nasir <[hidden email]>
Sent: Wednesday, 21 November 2012 10:05 PM
Subject: RE: measuring fluorescent intensity
 
Nurul,

I think the answer depends on whether you have calibrated your images. The documentation for Set Measurements at

http://rsbweb.nih.gov/ij/docs/menus/analyze.html#set

The mean gray value is the sum of gray values of all the pixels in the selection divided by the number of pixels [and additional notes on Analyze>Calibrate.]

And

"Integrated Density - Calculates and displays two values: "IntDen" (the product of Area and Mean Gray Value) and "RawIntDen" (the sum of the values of the pixels in the image or selection). "RawIntDen" is only available in ImageJ 1.44c or later. "IntDen" and "RawIntDen" values are the same for uncalibrated image. The Dot Blot Analysis example demonstrates how to use this option to analyze a dot blot assay. " [But if the image is calibrated Area does not equal pixels, thus IntDen does not equal RawIntDen.]

Charles

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Nurul Alimah Abdul Nasir
Sent: Wednesday, November 21, 2012 6:27 AM
To: [hidden email]
Subject: measuring fluorescent intensity


Hi there,
 
I want to measure the fluorescence of dye in various part of eye. I'm attaching examples of my pictures. (Tv79, I want to measure intensity of whole picture, while in TV104, I want to measure selected structure only) However, I am very confused using Image J. Should I take the int. Density measurement or mean gray area?
Is
the measurement is affected by how large the area of cell/tissue? If so, this means I need to standardize area size in all my sample ( which is also another problem as I'm not able to do that..)
 
Can anyone
care to guide me step by step way in measuring intensity for my case?
(I'm currently only using technique as written in this blog : http://sciencetechblog.com/2011/05/24/measuring-cell-fluorescence-using-imagej/ )
 
If anyone can guide me, it is very helpful. Thank you everyone in advance
 
Dr. Nurul Alimah Abdul Nasir
MBBS (Malaya)
Trainee Lecturer, Pharmacology, FOM UiTM
[phone]
[phone]

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

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

[nurul]

Dear Michael,

The image that I captured was based on the standard exposure time. Some of the picture doesn't have that high saturation. (by the way, what is the actual meaning of large area that are saturated? - i assume it is the high level of intensity of the colour.)

 
Dr. Nurul Alimah Abdul Nasir
MBBS (Malaya)
Trainee Lecturer, Pharmacology, FOM UiTM
[phone]
[phone]




________________________________
 From: "Cammer, Michael" <[hidden email]>
To: 'Nurul Alimah Abdul Nasir' <[hidden email]>
Sent: Wednesday, 21 November 2012 11:26 PM
Subject: RE: measuring fluorescent intensity
 
Hi.

The images you sent have large areas that are saturated.  Images need to be collected without saturated pixels to be properly analyzed.

Regards,

Michael

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Nurul Alimah Abdul Nasir
Sent: Wednesday, November 21, 2012 7:27 AM
To: [hidden email]
Subject: measuring fluorescent intensity


Hi there,
 
I want to measure the fluorescence of dye in various part of eye. I'm attaching examples of my pictures. (Tv79, I want to measure intensity of whole picture, while in TV104, I want to measure selected structure only) However, I am very confused using Image J. Should I take the int. Density measurement or mean gray area?
Is
the measurement is affected by how large the area of cell/tissue? If so, this means I need to standardize area size in all my sample ( which is also another problem as I'm not able to do that..)
 
Can anyone
care to guide me step by step way in measuring intensity for my case?
(I'm currently only using technique as written in this blog : http://sciencetechblog.com/2011/05/24/measuring-cell-fluorescence-using-imagej/ )
 
If anyone can guide me, it is very helpful. Thank you everyone in advance
 
Dr. Nurul Alimah Abdul Nasir
MBBS (Malaya)
Trainee Lecturer, Pharmacology, FOM UiTM
[phone]
[phone]

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

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

[Glen MacDonald-2]

Dear Nurul,
Saturation occurs when the detector cannot absorb any more photons.  2 things occur: since differences in signal describe structure, you cannot record structure in the saturated region.  The other is that you do not know the actual intensity for each saturated sample point, which makes quantitation irrelevant.   An important point is that there is no such thing as a standard exposure time  that can be applied for all specimens or experiments.    You must empirically determine an exposure that will not saturate your brightest specimen then apply that to all samples being compared.  The exposure time also needs to be tested on an unstained labeling control sample to check for autofluorescence, and tested on single-labeled positive controls (in the case of multiple labels) to test for bleed through.

the difference between mean and integrated intensity is an issue if you think the cells are changing volume.  If the volume stays constant, then mean intensity is the easiest value.  Integrated intensity normalizes to area, which is useful for cells or tissues that change volume.  Int.Den. is the mean intensity of the area times the area.   RawIntDen is the sum of intensities within the area, and may be better when the area has large variations of intensity.  

regards,
glen
Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]








On Nov 22, 2012, at 2:08 AM, Nurul Alimah Abdul Nasir wrote:

> Dear Michael,
>
> The image that I captured was based on the standard exposure time. Some of the picture doesn't have that high saturation. (by the way, what is the actual meaning of large area that are saturated? - i assume it is the high level of intensity of the colour.)
>
>  
> Dr. Nurul Alimah Abdul Nasir
> MBBS (Malaya)
> Trainee Lecturer, Pharmacology, FOM UiTM
> [phone]
> [phone]
>
>
>
>
> ________________________________
> From: "Cammer, Michael" <[hidden email]>
> To: 'Nurul Alimah Abdul Nasir' <[hidden email]>
> Sent: Wednesday, 21 November 2012 11:26 PM
> Subject: RE: measuring fluorescent intensity
>
> Hi.
>
> The images you sent have large areas that are saturated.  Images need to be collected without saturated pixels to be properly analyzed.
>
> Regards,
>
> Michael
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of Nurul Alimah Abdul Nasir
> Sent: Wednesday, November 21, 2012 7:27 AM
> To: [hidden email]
> Subject: measuring fluorescent intensity
>
>
> Hi there,
>  
> I want to measure the fluorescence of dye in various part of eye. I'm attaching examples of my pictures. (Tv79, I want to measure intensity of whole picture, while in TV104, I want to measure selected structure only) However, I am very confused using Image J. Should I take the int. Density measurement or mean gray area?
> Is
> the measurement is affected by how large the area of cell/tissue? If so, this means I need to standardize area size in all my sample ( which is also another problem as I'm not able to do that..)
>  
> Can anyone
> care to guide me step by step way in measuring intensity for my case?
> (I'm currently only using technique as written in this blog : http://sciencetechblog.com/2011/05/24/measuring-cell-fluorescence-using-imagej/ )
>  
> If anyone can guide me, it is very helpful. Thank you everyone in advance
>  
> Dr. Nurul Alimah Abdul Nasir
> MBBS (Malaya)
> Trainee Lecturer, Pharmacology, FOM UiTM
> [phone]
> [phone]
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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

[alemsqdt]

Hello all, I hope you have solved your problem (for the post is quite old) but in case you haven't, I tell you that I had the same problem (including the saturation fact) and I found the answer here: https://digital.bsd.uchicago.edu/resources_files/basic%20image%20quantification.pdf
(if you can't get the archive you can write to me and I will send it to you by e-mail)

About this
"An important point is that there is no such thing as a standard exposure time  that can be applied for all specimens or experiments.    You must empirically determine an exposure that will not saturate your brightest specimen then apply that to all samples being compared."

I agree It would be perfect to have "unsaturated" images, but I think that having saturated images is quite common in the real (not ideal) world. That's because you never know exactly which is the maximum expected intensity before doing the experiment (that's why youŕe doing the experiment!) so you have to guess it (using a few of your samples, and some theoretical background) and based on that information you set the "standard" conditions of the microscope which you must maintain constant in order to be able to compare the values between images thereafter. In other words, it's imposible to "determine an exposure that will not saturate your brightest specimen" because you don't know already which is your brightest specimen.

So, if you have a sample with more intensity than you have expected at the begining of the experiment you can not modify again the setup. But I would never say that if "you do not know the actual intensity for each saturated sample point, which makes quantitation irrelevant". In my case (and maybe in yours too) it's enough to say that there is more intensity in one tissue than in another and you can consider the saturated value as a maximum and compare it with the unsaturated area without problems (in the way described in the pdf)

Hope it helps

Regards!

Alejandro Serrano