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:
Identify primary Automatic
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?