Identify secondary objects nuclei and cytoplasm

identifysecondary
nuclei
neuron
cytoplasm

#1

Hi,

I am new to CelProfiler and I am trying to create a pipeline to identify nuclear stains and cytoplasmic staining on neurons based on DAPI staining to identify the primary objects and I am unsure how best to proceed with identifying secondary objects.

Any help appreciated,

Charlie


#2

Hi,

Do you mean you’d like to identify a whole neuron (nuclei + cytoplasm) based on DAPI staining ?

I’m a bit confused , may you specify a bit with some image?


#3

Hi,

I’d like to basically see if a neuron is positive for a marker (stained in
the image on another channel) both for nuclear and cytoplasmic proteins


#4

Thanks for the image.

You can try to use DAPI channel for IdentifyPrimaryObjects, name these objects “nuclei” for instance
and Green channel for IdentifySecondaryObjects, name these objects “cytoplasm” for instance.
In IdentifySecondaryObjects, try method “Distance - N” and adjust N to capture as much cytoplasm as possible. Neurons and dendrites are indeed very challenging to segment correctly.

After having identified objects (nucle, cytoplasm), you can then measure signal of interest as usual with MeasureObjectIntensity.


#5

Thanks. Is there also a way that I can get cell profiler to calculate
percentages of cells that are positive for a stain? Or would I need to be
setting my own limits for identifysecondaryobject to ensure only positive
cells and not background are recorded?


#6

Yes you can certainly use “FilterObjects” to set a selection threshold : Objects above the threshold will be kept as “positive”.
Eventually you will have a positive object count versus a total count , which then derives to the percentage you look for.