I took a look at your original pipeline and I think I identified some key problems that likely affected your ability to implement @bcimini’s recommendations.
My #1 best recommendation is to watch our Intro to CellProfiler tutorial on YouTube. This video tutorial will lay the groundwork for your image analysis task, to help you understand the principles of segmentation (in general and specifically within CellProfiler). It will also help you to understand how and why things can fail, which will make troubleshooting easier! And finally, it will show you how to access the help within CellProfiler, which details what each module does. Available here, on the COBA YouTube channel: CellProfiler Workshop - YouTube
Next, some specifics for your first pipeline:
Your CorrectIlluminationCalculate module creates an image where the pixel intensity values are > 1 because you’ve set “Rescale the illumination function” to “Yes” (this rescale operation rescales the pixel intensity values from 1 → infinity, see the help for more explanation about why). Below I’ve included a screenshot showing the CorrectIlluminationApply output image and a histogram showing that the values range from 1 to > 6:
As a result, when you invert your image in the following step using ImageMath, the output actually has pixel intensity values ranging 0 to -5:
Your IdentifyPrimaryObjects module was then set to use a manual threshold of 0 intensity. However, all of the pixels in your image have intensities < 0, which means that every pixel is identified has background. As a result, no objects are identified!
A few quick suggestions:
- My experiments suggest that an inversion can be performed on your original image
- This inverted image can be used within IdentifyPrimaryObjects
- An automated method for identifying the threshold would likely generalize to other images more consistently
- Adjusting the diameter to fit the typical diameter of your objects should help
- Finally, you may need to adjust the declumping parameters
Good luck and hope this helps! And don’t forget to watch the tutorial!