Hi I’m a complete beginner to this program. I played CountSpeckles pipeline to orient myself but I can’t seem to find the right parameters.
I have fluorescently labeled cells (Red and Blue with puncta-like staining pattern at times) in culture. The dye is a membrane incorporating dye (DiI and DiD). There may be a membrane exchange/fragment updake phenotype that seems to change with certain experimental treatments. Ideally I’d like to quantify the following:
- % of blue cells having red speckles inside (and vice versa).
- Number of speckles in the cells.
Some main problems I don’t know how to get around:
Non uniform puncta like stain makes it difficult to understand what color the cell was originally. By eye, you can tell blue or red cells mostly (cells with a uniform color tint or high population of a single color speckles). How to program the software to identify these correctly?
(related to first concern above) How can the software distinguish the periphery of the cell in a crowded region of interest? Feels like software will have hard time to enumerate cells
Example image I’m trying to analyze is the attached.