Quantify fluorescence intensity of the second parameter


I am trying to find appropriate settings to quantify the object intensity of fluorescent beads. Those are Spherotech beads with 8 different intensities but the same wavelength. Originally this analysis was performed with an imaging cytometer; pictures were saved in flf-mode.
After converting the pictures to 12-bit tif files I try the analysis with cellprofiler using the following settings:
Load images
Identify primary Automatic
Identify secondary
Measure object intensity

The primary is the transmission image where each bead shows the same or almost the same intensity. If the fluorescence images were used as the primary channel. the three populations with the lowest fluorescence intensities would simply not be identified.
The basic idea now is to find the x-y-position of all beads with transmission images and subsequently quantify the intensity of those positions on the second set of pictures. But apparently object intensity is not quantified for the second parameter…is that possible? Does anybody know how to deal with that kind of analysis?


Hi Dominik,

After you identify the beads with IdentifyPrimAutomatic, you should not need to use the IdentifySecondary module. This module creates secondary objects around the primary objects.

I think what you want to do is use MeasureObjectIntensity on your primary objects, but using the fluorescent image. Does this make sense? Then you can export all of the primary object data, and it will give you the x-y locations as well as the integrated and mean intensity of each bead in the fluorescent channel. Please ask if you cannot get this working!