Time-lapse live cell imaging of a growing cell population is routine in many biological investigations. A major challenge in imaging analysis is accurate segmentation, a process to define the boundaries of cells based on raw image data. Current segmentation methods relying on single boundary features have problems in robustness when dealing with inhomogeneous foci which invariably happens in cell population imaging. Here, we demonstrated that combined with multi-layer training set strategy, a neural-network-based algorithm Cellbow can achieve accurate and robust segmentation of cells in broad and general settings. It can also facilitate long-term tracking of cell growth and division. Furthermore, Cellbow is customizable and generalizable. It is broadly applicable to segmenting fluorescent images of diverse cell types with no further training needed. For bright- field images, only a small set of sample images of the specific cell type from the user may be needed for training. To facilitate the application of Cellbow, we provide a website on which one can online test the software, as well as an ImageJ plugin for the user to visualize the performance before software installation.### Competing Interest StatementThe authors have declared no competing interest.

Cellbow: a robust customizable cell segmentation program