I was hoping someone might be able to help a little with segmentation on these images. Briefly this is an antiviral assay - cells were grown in 96 well plates and treated with different antiviral drugs prior to infection.
Cells were fixed, stained with DAPI / cell mask blue to outline nucleus / cytoplasm (both same fluoresence - I am more interested in being able to outline the whole cell) and then stained with antiviral antibody / alexa555 secondary. Goal of the assay is viral antibody expression between treatments in terms of the number of cells infected / intensity of staining.
Segementation of uninfected cells is not brilliant but ok (image 1). Unfortunately the infected cells fuse to produce large multinucleated cells which makes segmentation difficult (see image 2) and to make it more difficult the nuclei are saturated. Is there a way to segment these multinucliated cells so that each nuclei is counted, even if the cytoplasm allocated to that cell is not accurate - that would at least enable calculations of infected versus uninfected cells. Any other thoughts as to how quantify? Integrated intensity?
I have attached my pipeline. Any assistance greatly appreciated.
Thanks in advance
130707 final pipeline.cp (13.3 KB)