Object Research Systems 1 article published in JoVE Biology Deep Learning-Based Segmentation of Cryo-Electron Tomograms Jessica E. Heebner1, Carson Purnell1, Ryan K. Hylton1, Mike Marsh2, Michael A. Grillo1, Matthew T. Swulius1 1Pennsylvania State University-College of Medicine, 2Object Research Systems This is a method for training a multi-slice U-Net for multi-class segmentation of cryo-electron tomograms using a portion of one tomogram as a training input. We describe how to infer this network to other tomograms and how to extract segmentations for further analyses, such as subtomogram averaging and filament tracing.