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Whole-cell segmentation of tissue images with human-level performance using large-scale data annotation and deep learning

Nature Biotechnology volume 40, pages 555–565 (2022)Cite this article

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Abstract

A principal challenge in the analysis of tissue imaging data is cell segmentation—the task of identifying the precise boundary of every cell in an image. To address this problem we constructed TissueNet, a dataset for training segmentation models that contains more than 1 million manually labeled cells, an order of magnitude more than all previously published segmentation training datasets. We used TissueNet to train Mesmer, a deep-learning-enabled segmentation algorithm. We demonstrated that Mesmer is more accurate than previous methods, generalizes to the full diversity of tissue types and imaging platforms in TissueNet, and achieves human-level performance. Mesmer enabled the automated extraction of key cellular features, such as subcellular localization of protein signal, which was challenging with previous approaches. We then adapted Mesmer to harness cell lineage information in highly multiplexed datasets and used this enhanced version to quantify cell morphology changes during human gestation. All code, data and models are released as a community resource.

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Fig. 1: A human-in-the-loop approach enables scalable, pixel-level annotation of large image collections.
Fig. 2: Mesmer delivers accurate nuclear and whole-cell segmentation in multiplexed images of tissues.
Fig. 3: Mesmer performs whole-cell segmentation across tissue types and imaging platforms with human-level accuracy.
Fig. 4: Mesmer enables accurate analysis of multiplex imaging data.
Fig. 5: Lineage-aware segmentation enables morphological profiling of cells in the decidua during human pregnancy.
Fig. 6: Cloud-native and on-premise software facilitates deployment of Mesmer.

Data availability

The TissueNet dataset is available at https://datasets.deepcell.org/ for noncommercial use.

Code availability

All software for dataset construction, model training, deployment and analysis is available on our github page https://github.com/vanvalenlab/intro-to-deepcell. All code to generate the figures in this paper is available at https://github.com/vanvalenlab/publication-figures/tree/master/2021-Greenwald_Miller_et_al-Mesmer.

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Acknowledgements

We thank K. Borner, L. Cai, M. Covert, A. Karpathy, S. Quake and M. Thomson for interesting discussions; D. Glass and E. McCaffrey for feedback on the manuscript; T. Vora for copy editing; R. Angoshtari, G. Barlow, B. Bodenmiller, C. Carey, R. Coffey, A. Delmastro, C. Egelston, M. Hoppe, H. Jackson, A. Jeyasekharan, S. Jiang, Y. Kim, E. McCaffrey, E. McKinley, M. Nelson, S.-B. Ng, G. Nolan, S. Patel, Y. Peng, D. Philips, R. Rashid, S. Rodig, S. Santagata, C. Schuerch, D. Schulz, Di. Simons, P. Sorger, J. Weirather and Y. Yuan for providing imaging data for TissueNet; the crowd annotators who powered our human-in-the-loop pipeline; and all patients who donated samples for this study. This work was supported by grants from the Shurl and Kay Curci Foundation, the Rita Allen Foundation, the Susan E. Riley Foundation, the Pew Heritage Trust, the Alexander and Margaret Stewart Trust, the Heritage Medical Research Institute, the Paul Allen Family Foundation through the Allen Discovery Centers at Stanford and Caltech, the Rosen Center for Bioengineering at Caltech and the Center for Environmental and Microbial Interactions at Caltech (D.V.V.). This work was also supported by 5U54CA20997105, 5DP5OD01982205, 1R01CA24063801A1, 5R01AG06827902, 5UH3CA24663303, 5R01CA22952904, 1U24CA22430901, 5R01AG05791504 and 5R01AG05628705 from NIH, W81XWH2110143 from DOD, and other funding from the Bill and Melinda Gates Foundation, Cancer Research Institute, the Parker Center for Cancer Immunotherapy and the Breast Cancer Research Foundation (M.A.). N.F.G. was supported by NCI CA246880-01 and the Stanford Graduate Fellowship. B.J.M. was supported by the Stanford Graduate Fellowship and Stanford Interdisciplinary Graduate Fellowship. T.D. was supported by the Schmidt Academy for Software Engineering at Caltech.

Author information

Author notes

  1. Cole Pavelchek

    Present address: Washington University School of Medicine in St. Louis, St. Louis, MO, USA

  2. Sunny Cui

    Present address: Department of Computer Science, Princeton University, Princeton, NJ, USA

  3. These authors contributed equally: Noah F. Greenwald, Geneva Miller.

Authors and Affiliations

  1. Cancer Biology Program, Stanford University, Stanford, CA, USA

    Noah F. Greenwald, Brianna J. McIntosh & Ke Xuan Leow

  2. Department of Pathology, Stanford University, Stanford, CA, USA

    Noah F. Greenwald, Alex Kong, Adam Kagel, Christine Camacho Fullaway, Ke Xuan Leow, Jaiveer Singh, Mara Fong, Gautam Chaudhry, Zion Abraham, Jackson Moseley, Shiri Warshawsky, Erin Soon, Shirley Greenbaum, Tyler Risom, Sean C. Bendall & Michael Angelo

  3. Division of Biology and Bioengineering, California Institute of Technology, Pasadena, CA, USA

    Geneva Miller, Erick Moen, Thomas Dougherty, Morgan Sarah Schwartz, Cole Pavelchek, Isabella Camplisson, William Graf & David Van Valen

  4. Department of Electrical Engineering, California Institute of Technology, Pasadena, CA, USA

    Sunny Cui

  5. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

    Omer Bar-Tal & Leeat Keren

  6. Department of Cognitive, Linguistic and Psychological Sciences, Brown University, Providence, RI, USA

    Mara Fong

  7. Immunology Program, Stanford University, Stanford, CA, USA

    Erin Soon

  8. Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Travis Hollmann

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  1. Noah F. Greenwald
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Contributions

N.F.G., L.K., M.A. and D.V.V. conceived the project. E.M. and D.V.V. conceived the human-in-the-loop approach. L.K. and M.A. conceived the whole-cell segmentation approach. G.M., T.D., E.M., W.G. and D.V.V. developed DeepCell Label. G.M., N.F.G., E.M., I.C., W.G. and D.V.V. developed the human-in-the-loop pipeline. M.S.S., C.P., W.G. and D.V.V. developed Mesmer’s deep learning architecture. W.G., N.F.G. and D.V.V. developed model training software. C.P. and W.G. developed cloud deployment. M.S.S., S.C., W.G. and D.V.V. developed metrics software. W.G. developed plugins. N.F.G., A. Kong, A. Kagel, J.S. and O.B.-T. developed the multiplex image analysis pipeline. A. Kagel and G.M. developed the pathologist evaluation software. N.F.G., G.M. and T.H. supervised training data creation. N.F.G., C.C.F., B.J.M., K.X.L., M.F., G.C., Z.A., J.M. and S.W. performed quality control on the training data. E.S., S.G. and T.R. generated MIBI-TOF data for morphological analyses. S.C.B. helped with experimental design. N.F.G., W.G. and D.V.V. trained the models. N.F.G., W.G., G.M. and D.V.V. performed data analysis. N.F.G., G.M., M.A. and D.V.V. wrote the manuscript. M.A. and D.V.V. supervised the project. All authors provided feedback on the manuscript.

Corresponding authors

Correspondence to Michael Angelo or David Van Valen.

Ethics declarations

Competing interests

M.A. is an inventor on patent US20150287578A1. M.A. is a board member and shareholder in IonPath Inc. T.R. has previously consulted for IonPath Inc. D.V.V and E.M. have filed a provisional patent for this work. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 DeepCell Label annotation workflow.

a, How multichannel images are represented and edited in DeepCell Label. b, Scalable backend for DeepCell Label that dynamically adjusts required resources based on usage, allowing concurrent annotators to work in parallel. c, Human-in-the-loop workflow diagram. Images are uploaded to the server, run through Mesmer to make predictions, and cropped to facilitate error correction. These crops are sent to the crowd to be corrected, stitched back together, run through quality control to ensure accuracy, and used to train an updated model.

Extended Data Fig. 2 Mesmer benchmarking.

a, PanopticNet architecture. Images are fed into a ResNet50 backbone coupled to a feature pyramid network. Two semantic heads produce pixel-level predictions. The first head predicts whether each pixel belongs to the interior, border, or background of a cell, while the second head predicts the center of each cell. b, Relative proportion of preprocessing, inference, and postprocessing time in PanopticNet architecture. c, Evaluation of precision, recall, and Jaccard index for Mesmer and previously published models (right) and models trained on TissueNet (left). d, Summary of TissueNet accuracy for Mesmer and selected models to facilitate future benchmarking efforts e,f Breakdown of most prevalent error types (e) and less prevalent error types (f) for Mesmer and previously published models illustrates Mesmer’s advantages over previous approaches. g, Comparison of the size distribution of prediction errors for Mesmer (left) with nuclear segmentation followed by expansion (right) shows that Mesmer’s predictions are unbiased.

Extended Data Fig. 3 TissueNet accuracy comparisons.

a, Accuracy of specialist models trained on each platform type (rows) and evaluated on data from other platform types (columns) indicates good agreement within immunofluorescence and mass spectrometry-based methods, but not across distinct methods. b, Accuracy of specialist models trained on each tissue type (rows) and evaluated on data from other tissue types (columns) demonstrates that models trained on only a single tissue type do not generalize as well to other tissue types. c, Quantification of F1 score as a function of the size of the dataset used for training. d-h, Quantification of individual error types as a function of the size of the dataset used for training. i, Representative images where Mesmer accuracy was poor, as determined by the image specific F1 score. j, Impact of image blurring on model accuracy. k, Impact of image downsampling and then upsampling on model accuracy. l, Impact of adding random noise to image on model accuracy. All scale bars are 50 μM.

Extended Data Fig. 4 3D segmentation.

Proof of principle for using Mesmer’s segmentation predictions to generate 3D segmentations. A z-stack of 3D data is fed to Mesmer, which generates separate 2D predictions for each slice. We computationally link the segmentations predictions from each slice to form 3D objects. This approach can form the basis for human-in-the-loop construction of training data for 3D models.

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Greenwald, N.F., Miller, G., Moen, E. et al. Whole-cell segmentation of tissue images with human-level performance using large-scale data annotation and deep learning. Nat Biotechnol 40, 555–565 (2022). https://doi.org/10.1038/s41587-021-01094-0

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