Robust Optical Flow Algorithm for General, Label-free Cell Segmentation

Cell segmentation is crucial to the field of cell biology, as the accurate extraction of cell morphology, migration, and ultimately behavior from time-lapse live cell imagery are of paramount importance to elucidate and understand basic cellular processes. Here, we introduce a novel segmentation approach centered around optical flow and show that it achieves robust segmentation by validating it on multiple cell types, phenotypes, optical modalities, and in-vitro environments without the need of labels. By leveraging cell movement in time-lapse imagery as a means to distinguish cells from their background and augmenting the output with machine vision operations, our algorithm reduces the number of adjustable parameters needed for optimization to two. The code is packaged within a MATLAB executable file, offering an accessible means for general cell segmentation typically unavailable in most cell biology laboratories. ### Competing Interest Statement The authors have declared no competing interest.