I’ve been experimenting with using CellProfiler to automate our FFA (fluorescent focus assay) cell counting. In this assay, GFP-labelled antibodies attach to the surface of cells if certain virus genes are expressed in the cells. From the examples, it seems like the best way to count the cells is to use a DAPI stain first, but we’re kind of locked in to a test method without it. Thus, I’m trying to use the IdentifyPrimAutomatic to detect cells by their membranes, not their nucleuses.
Among the options for IdentifyPrimAutomatic, I noticed a Laplacian of Gaussian (LoG) counting option.
The inputs listed are size of neighborhood (height, width), Sigma, Minimum Area, Size for Weiner Filter (height, width), Threshold.
There is no explanation for this function or its variables within the help function. I assume Sigma is the analog of the Sigma for the LoG edge-finding function, and that threshold is the same as elsewhere, but I am unsure of how to handle the other variables (neighborhood size, minimum area, weiner filter size). For a starting point for those variables, should I use some function of object size, or average distance between objects, or what?