First time poster here.
I’d like to reduce my error rate when trying to automatically count cells with the Analyze Particles function.
Each time I binarize the photo using Adjust Threshold, I tend to lose some cells.
One issue I am aware of is that the photo quality is rather poor; namely, the lighting is uneven, so there are usually dark spots shading some cells, which means that I either lose the cells in that shady patch or have a large black blotch with poorly defined cells within it. If I then Fill Holes, for example, sometimes this whole blotch is filled, losing a number of cells. The Watershed function isn’t always enough to define the cells and retrieve that data.
Moreover, the binarize function creates a fair bit of noise in the form of these blotchy patches of black, which cannot easily be removed with the Remove Outliers function, without simultaneously losing some cells. When, in these instances, I Fill Holes and Watershed, these blotches often get divided up and create a number of false positives when I Analyze Particles. I tried playing around with raising circularity to as high as 0.7-1.00 because these blotches tend not to be particularly round, although that has not totally solved my issue.
I take some consolation in the fact that these two tendencies may somewhat balance each other out, as my loss of data may be somewhat accounted for by my false positives, but I’d still like to do better. I do not need 100% accuracy. I’m only trying to get fairly good estimates, so anything I can do reduce my error would be great.
Any suggestions for how I may lose less data and limit false positives?