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In this episode, Erin and Beth use CellProfiler to segment nuclei and cells in images that are difficult because the cells are all up in each other's business causing focus issues. We start with a threshold correction factor that is causing problems by being too low, deal with out-of-focus light by adding a block-based illumination correction, and end up using local thresholding. Erin advocates for just trashing the hard images and can't seem to count to 60 in her head. #science #openscience #microscopy #tutorial #opensource #biology #imageanalysis #segmentation #cellprofiler ================================================== On Ask Erin/Dear Beth, bioimage analysis experts Beth Cimini and Erin Weisbart, of the Imaging Platform at the Broad Institute of MIT and Harvard, answer your image analysis questions! Whether it’s about tuning or troubleshooting, segmenting or measuring, Beth and Erin have opinions and by golly are they going to share them! Using mostly CellProfiler, but with expertise in many other image analysis softwares such as ImageJ/FIJI, Cellpose, Piximi, and more, no image analysis question is too hard, so ask away! Submit your own questions at broad.io/AskErinDearBeth Images and pipelines we’ve used are available for you to download and play with at https://ciminilab.github.io/AskErin_D...