Measureobjectintensity function

cellprofiler-analyst

#1

Hello. I am staining cells with DAPI and alexa 488 to measure nuclei and cytoplasm fluorescence respectively. I have imported the image and used a pipeline which works perfectly, thank you cell profiler! My question is I get multiple excel sheet with “image”, “cytoplasm”, “nuclei” and “cell” with different intensity measurements for nuclei and cytoplasm. I have read through the forum and I don’t quite understand why there is a difference and which one I am supposed to use for measurement.

for the cytoplasm, if the cell has subtracted nuclei, why is there still intensity measurement for the dapi channel?

thank you.


#2

Uploading… I am trying to upload my images and pipeline but its taking a while, i don’t know why


#3

We seem to have an uploading problem right now, since I can’t fully upload either. We’ll check into that.

As for your science question, measurements are either Per_Image (e.g. the “Image” sheet) or Per_Object (e.g. “cytoplasm”, and any other objects you defined in the pipeline). Per_Image measurements are any measurements defined across the whole image, like image thresholds or object counts. Object measurements in the Per_Object tables are measurements across each individual object, like area or that object’s intensity. So it’s up to you to determine what measurement you want to analyze. You say you want “to measure nuclei and cytoplasm fluorescence”, then I assume you want to use MeasureObjectIntensity and then look in the nucleus and cytoplasm spreadsheets for the, say, mean intensity columns. You can average them per image or across the whole experiment depending on your protocol.

You should think of objects like nucleus and cytoplasm separately from the channels that typically define them. You might only care about the DAPI intensity in the nucleus object and the alexa intensity in the cytoplasm, but one can certainly measure the DAPI channel intensity in the cytoplasm and vice versa, though you might not care about this somewhat non-intuitive measure. Maybe there will even be some translocation that you didn’t expect and then you could measure the intensity change. The point is, you can measure any channel’s properties in any defined object. Hope that helps!


#4

yes, that was brilliant! thank you!


#5

FYI, the upload issue has been fixed, but no need to upload if you are satisfied now.


#6

For clarification, if you wanted to graph the intensity of the DAPI versus the intensity of the Alexa, you would use the values from the ‘DAPI’ spreadsheet, Intensity_MeanIntensity_DAPI, versus the values from the ‘Alexa’ spreadsheet, Intensity_MeanIntensity_Alexa?

I’m not sure I understand how the ‘DAPI’ object can have a Intensity_MeanIntensity_Alexa if the TIFF in which the DAPI object was defined was only representing the DAPI image. What are those values representing exactly? I don’t know if I can explain this correctly…but is it the Alexa object (so it’s size and shape) overlayed on the DAPI image…is that what Intensity_MeanIntensity_Alexa in the ‘DAPI’ channel would mean? Thanks for your help! @David_Logan @CellProfilerTeam


#7

You’re right that objects are defined in only one channel, but in MeasureObjectIntensity you have the option to measure them in any channel; imagine the case in your example above where your Alexa is on a nuclear marker, and you want to measure its brightness in the nucleus (as defined by DAPI). Whatever object’s spreadsheet you go to will tell you what object is being measured (it therefore helps to give your objects different names than your channels to minimize confusion!), and the Intensity_MeanIntensity_Channel is that object’s intensity in that channel.

Does that help at all?


#8

YES! Thank you so much. This was the little nudge my brain needed to fully understand what was going on! I really appreciate your help @bcimini