Re: measuring fluorescent intensity

Posted by nurul on Nov 21, 2012; 2:19pm
URL: http://imagej.273.s1.nabble.com/measuring-fluorescent-intensity-tp5000912p5000928.html

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]

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