Max Intensity values at 254 - maxing out?

Hi All,

We are following a fluorescent macromolecule uptake into lysosomes with using Integrated Density per cell.
At early times we have intensity values of Mean, Min, and Max such as 69, 27, and 190. And as time progresses these values increase, as expected.

But at the latest times, most of these values have Max values of 254, such as Mean, Min, and Max of 157, 49, and 254, or as in 78, 23, and 254. While these Max values are below 255 which I understand to be the maximum intensity possible, will these consistent 254 Max values indicate a “maxing out” of fluorescent intensity?

Thanks to any help!

Hi @ClydeM,

If you can provide a small dataset representative of your experiment, we can probably help you figure out if it’s saturation or something else.


Hi Oli,

Thanks for the fast reply! OK - here is one set:

hrs Batch File Name Thrshold% Area Mean Min Max IntDen, millions #cells ID/cell
24 18A a-1 Mom=1.03 11657 138 57 254 1.61 13 123846
a-2 Mom=1.36 12,583 84 38 211 1.06 13 81538
a-3 Mom=1.29 10,247 109 30 254 1.12 9 124444
a-4 Mom=1.10 6460 105 36 254 0.678 5 135600
a-5 Mom=1.16 10395 125 39 254 1.3 12 108333
a-6 Mom=1.25 14365 157 49 254 2.26 17 132941
a-7 Mom=0.36 12276 139 45 254 1.71 12 142500
a-8 Mom=1.06 3139 146 34 254 0.458 3 152667
a-9 Ots=1.31 15582 161 49 254 2.51 10 251000
a-10 Mom=1.22 14658 124 36 254 1.82 15 121333
a-11 Man=2.09 11997 110 31 254 1.32 11 120000

Oh I’m sorry. By dataset I meant a couple of images…

I was busy today and did not notice your reply.


Hi Oli - No problem. Images make more sense too. Here is one:

a-9 80 500

Hi Oli,

Can you suggest a good way to copy the image? In my first reply, it seems the file is too large.

Good day,

your sample image is definitely over-exposed!

It comes as an RGB-image but contains only data in the red channel.
In fact the maximum value in the red channel is 254 and 3727 pixels bear this value. There are no pixels with value 255 and from this fact I conclude that the camera or other steps during image acquisition limit the range of values to 0…254.

Due to the cliiping this image is unsuited for serious image processing.



1 Like


I appreciate the rapid reply. I have a couple more questions. Can you tell me if the following image has this problem?

a-3 80 500

Also, Can you suggest an approach to account for a wide range of intensities that increase over time?

Thanks so much

Good day,

this sample image is not over-exposed, there is only one pixel having the value 254 and none having a value of 255.

Can you suggest an approach to account for a wide range of intensities that increase over time?

Not perfectly sure what you mean but 16bit images can handle the range 0…65535. However, you need a camera or opto-electronic device that is able to output 16bit images. For instance most modern scanner do and some cameras can output more than 8bit in raw format, i.e. they are able to deliver images in 16bit format although their signals are in fact 12 or 14bits deep.

Please study the manuals of your equipment and check all processing steps.




Very much appreciate your confirmation that this image was not over-exposed. While we now can judge by eye what is probably over-exposed, it is better to know for sure.
How did you determine the number of pixels with a given value, such as 254.

Thanks so much

How did you determine the number of pixels with a given value, such as 254.

Have a look at the histogram of yourimages and please consider looking into the ImageJ-userguide:




I am working my way through the userguide. But I have a question about comparing fluorescence intensities.
In one image of cells our agent accumulates in the nucleus (large area of the cell). In another image our modified agent accumulates in lysosomes (bright punctate and small area) of the same cell line. Is it appropriate to compare the integrated density per cell of these two images as an indicator of greater cell content of the agent?

Thanks so much.

Hello ClydeM,
If I understand you correctly the amount of intensity increase over time IS the amount of uptake over time. Am I correct?

Hello Robert

Well, that is the question. Could other factors cause the increased intensity, such as smaller volumes, binding to membranes, or self complexation?