Counting with hemocytometer


I am trying to build a pipeline for images of of E. coli cells in a hemocytometer chamber. I would like be able to count the number of cells, however have been unable to identify cells that are overlapping the grid lines of the hemocytometer or are near the edge. Parts of the gridlines are also counted sometimes. After reading through many forums, the gridlines do not seem to be the only problem as my cells are extremely small, and dark with a halo of light around them while the grid lines have a much higher intensity. I tried to enhance the cells by using the enhance or suppress features module, enhance dark circles.
I am new to Cell Profiler so any advice on what to do next would be greatly appreciated. I have attached one of the images and the pipeline I have come up with so far.

E.coliHemocytometer.mat (52.3 KB)


I’m attaching a pipeline which I think may help; it’s not too far off from your current attempt, I think. I removed the portion that crops the image; I don’t know if there’s a way to obtain the relevant region without manual intervention so I will leave that part up to you.

2013_01_25.cp (6.42 KB)