I am trying to analyze images that contain a heterogeneous population of Drosophila blood cells (hemocytes). The majority of the cells are round and vary in size from ~10-40 um diameter (these are plasmatocytes or activated plasmatocytes). There are some larger cells with a banana-like shape that are often 5x larger than the plasmatocytes (these are lamelloocytes).
My images start off as .nd2 files. I have been converting Z-stack projection images from each of the three channels (I have a nuclear marker as one of my channels) into 16-bit grayscale images and them saving them as .TIF files for CP analysis. I would ultimately like to be able to use CP to extract the following general information:
- Cell size, shape (i.e. spikiness)
- Intensity for each channel per cell
- % nuclear localization of certain proteins (labeled as channel 2 in attached images)
- BONUS: number and size of puncta per cell
Attached below are sample images (one from each channel) and the modified pipeline that I have been working with.
Here are my questions:
The cells are stained with phalloidin (red channel) so that I can see the outline of each cell. The control cells stain much less strongly for phalloidin than the mutant cells. Therefore, in order to see the outline of the control cells, I have to crank up the contrast much higher than I do for the mutant cells. I have been adjusting the contrast for my 16-bit images in FIJI prior to saving them as .TIF files for CP. Thankfully, adjusting the contrast in FIJI does not appear to change the intensity readout from CP. However is there a way to make these contrast adjustments directly in CP?
The CP pipeline that I am using is doing a reasonable job of identifying the round cells (plasmatocytes) but it is chunking up the larger banana-like cells (lamellocytes) into multiple smaller cells (even though there is only one nuclei for most of these large banana-shaped cell). Any ideas about how I could tweak my pipeline so that I can simultaneously analyze both cell types in my images?
Some of the cells are multinucleate. How do I get cell profiler to recognize that two or more nuclei belong to one cell?
Thanks so much for any help that you can provide! I look forward to using CP for my analysis.
Basic analysis.cpproj (537 KB)