http://imagej.273.s1.nabble.com/measuring-fluorescent-intensity-tp5000912p5000949.html
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.
> 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
> +603-55211298
> +6013-2064397
>
>
>
>
> ________________________________
> 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
> +603-55211298
> +6013-2064397
>
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