Wrong Intensities Measurements

cellprofiler

#1

Hi,
I have question about inensities measurement. I am measuring Alexa intensity in cell mask from GFP channel. The images are 12-bit TIF saved as 16-bit.
So I am doing rescaling E (min/max) But Ijust found that the inensities measurements do not correspond to the image intensities.
In Alexa image I have some saturated pixels (rescaled intensity 1), the pixels are within object mask, but in the output table my max intensity
is 0.6. Could you help me?
I also found that there is something strange in rescale intensity module help for method E - settings 0, 0.0625, 0, 0, 1, 1. For me it did not work, I had to
se 0, 0.0625, 0, 1, 0, 1. Is it correct?
Thanks a lot.
Milan
I am using Windows XP, Cell Profile version 1.0.6031.

P.S. I could attached only pipeline and two .zip compressed pictures, so GFP picture is missing. I can send it separately.
DAPI - n000000.tif.zip (1.38 MB)
Alexa 647 - n000000.tif.zip (1.85 MB)
tubulin3.pipe.mat (1.45 KB)


#2

Hi,

You are correct about the help for that module- I’ve never noticed it, but it says

[quote] To convert 12-bit images saved in 16-bit format to the correct
range, use the settings 0, 0.0625, 0, 0, 1, 1. The value 0.0625 is equivalent to 2^12 divided by 2^16, so it will convert a 16 bit image containing only 12 bits of data to the proper range. [/quote]

It should say

[quote] To convert 12-bit images saved in 16-bit format to the correct
range, use the settings 0, 0.0625, 0, 1, 0, 1. The value 0.0625 is equivalent
to 2^12 divided by 2^16, so it will convert a 16 bit image containing
only 12 bits of data to the proper range. [/quote]

As for the intensity measurement, I do see some pixels that look almost saturated (0.9998) that look like they would be within a cell mask. I made an arbitrary mask which contained these pixels (since I do not have the GFP image), and used MeasureObjectIntensity. Looking at Intensity_MaxIntensity_RescaledBlue, I do get 0.9998 for that object. Double check that you are using MeasureObjectIntensity and that you’re looking at the MaxIntensity measurement for the object of interest. When you say ‘output table’ do you mean the Excel file? If you only output ‘Image’, this will be the average of all the objects in your image, so it could very well be 0.6.

~kate


#3

Hi Kate,
thank you for your fast answer. Now I see, so this parameter is not Max Intensity of pixel from all segmented mask, but mean of max. intensity pixels
from all objects.
Thanks a lot.
Milan