Threshold/count cytosolic stained cells

I would appreciate any input in what is the best way to count these cells. All threshold methods I have tried and filters are making me loose a lot of cells. What is the best way to do this? I have a Dapi chanel also, but I need to get a lymphocyte count vs. all cells count ratio.
lymphocytes_Crop_Composite_5Dec16.zip (1.7 MB)

lymphocytes_Crop_Composite_5Dec16.zip (1.7 MB)

Having played with bkground subtraction and blurring, It seems you may need better resolution. also with cells piling up on eachother it creates the blur effect, you could try thresolding an ROI without cells toppled on top of eachother.

DAPI should be easy to identify total cells and you may need a lypmphocyte marker to gate your populations.

Sorry I couldn’t help further!

Darren

Hi @alice

Sometimes it helps to get a segmentation from Dapi and then refine (exclude) cells which are not present in the other channel.

Is the Dapi-channel already part of the uploaded tif?

No. I will upload the Dapi chanel next, and maybe you can guide me thru the process? Many Thanks!

Dapi_Crop_Composite_5Dec16.zip (1.8 MB)