1. Computational and Systems Biology
  2. Neuroscience
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On the objectivity, reliability, and validity of deep learning enabled bioimage analyses

  1. Dennis Segebarth
  2. Matthias Griebel
  3. Nikolai Stein
  4. Cora R von Collenberg
  5. Corinna Martin
  6. Dominik Fiedler
  7. Lucas B Comeras
  8. Anupam Sah
  9. Victoria Schoeffler
  10. Teresa Lüffe
  11. Alexander Dürr
  12. Rohini Gupta
  13. Manju Sasi
  14. Christina Lillesaar
  15. Maren D Lange
  16. Ramon O Tasan
  17. Nicolas Singewald
  18. Hans-Christian Pape
  19. Christoph M Flath  Is a corresponding author
  20. Robert Blum  Is a corresponding author
  1. University Hospital Würzburg, Germany
  2. University of Würzburg, Germany
  3. Westf?lische Wilhlems-Universit?t, Germany
  4. University of Innsbruck, Austria
  5. University of Inssbruck, Austria
Research Article
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Cite this article as: eLife 2020;9:e59780 doi: 10.7554/eLife.59780

Abstract

Bioimage analysis of fluorescent labels is widely used in the life sciences. Recent advances in deep learning (DL) allow automating time-consuming manual image analysis processes based on annotated training data. However, manual annotation of fluorescent features with a low signal-to-noise ratio is somewhat subjective. Training DL models on subjective annotations may be instable or yield biased models. In turn, these models may be unable to reliably detect biological effects. An analysis pipeline integrating data annotation, ground truth estimation, and model training can mitigate this risk. To evaluate this integrated process, we compared different DL-based analysis approaches. With data from two model organisms (mice, zebrafish) and five laboratories, we show that ground truth estimation from multiple human annotators helps to establish objectivity in fluorescent feature annotations. Furthermore, ensembles of multiple models trained on the estimated ground truth establish reliability and validity. Our research provides guidelines for reproducible DL-based bioimage analyses.

Article and author information

Author details

  1. Dennis Segebarth

    Institute of Clinical Neurobiology, University Hospital Würzburg, Würzburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3806-9324
  2. Matthias Griebel

    Department of Business and Economics, University of Würzburg, Würzburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1959-0242
  3. Nikolai Stein

    Department of Business and Economics, University of Würzburg, Würzburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Cora R von Collenberg

    Institute of Clinical Neurobiology, University Hospital Würzburg, Würzburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Corinna Martin

    Institute of Clinical Neurobiology, University Hospital Würzburg, Würzburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Dominik Fiedler

    Institute of Physiology, Westf?lische Wilhlems-Universit?t, Münster, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Lucas B Comeras

    Department of Pharmacology, University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  8. Anupam Sah

    Department of Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  9. Victoria Schoeffler

    Department of Child and Adolescent Psychiatry, University Hospital Würzburg, Würzburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Teresa Lüffe

    Department of Child and Adolescent Psychiatry, University Hospital Würzburg, Würzburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  11. Alexander Dürr

    Department of Business and Economics, University of Würzburg, Würzburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  12. Rohini Gupta

    Institute of Clinical Neurobiology, University Hospital Würzburg, Würzburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  13. Manju Sasi

    Institute of Clinical Neurobiology, University Hospital Würzburg, Würzburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  14. Christina Lillesaar

    Department of Child and Adolescent Psychiatry, University Hospital Würzburg, Würzburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5166-2851
  15. Maren D Lange

    Institute of Physiology, Westf?lische Wilhlems-Universit?t, Münster, Germany
    Competing interests
    The authors declare that no competing interests exist.
  16. Ramon O Tasan

    Department of Pharmacology, University of Inssbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
  17. Nicolas Singewald

    Department of Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0166-3370
  18. Hans-Christian Pape

    Institute of Physiology, Westf?lische Wilhlems-Universit?t, Münster, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6874-8224
  19. Christoph M Flath

    Department of Business and Economics, University of Würzburg, Würzburg, Germany
    For correspondence
    christoph.flath@uni-wuerzburg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1761-9833
  20. Robert Blum

    Institute of Clinical Neurobiology, University Hospital Würzburg, Würzburg, Germany
    For correspondence
    Blum_R@ukw.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5270-3854

Funding

Deutsche Forschungsgemeinschaft (ID 44541416 - TRR58,A10)

  • Robert Blum

Deutsche Forschungsgemeinschaft (ID 44541416 - TRR58,A03)

  • Hans-Christian Pape

Deutsche Forschungsgemeinschaft (ID 44541416 - TRR58,B08)

  • Maren D Lange

Graduate School of Life Sciences Wuerzburg (fellowship)

  • Rohini Gupta
  • Manju Sasi

Austrian Science Fund (P29952 & P25851)

  • Ramon O Tasan

Austrian Science Fund (I2433-B26,DKW-1206,SFB F4410)

  • Nicolas Singewald

Interdisziplinaeres Zentrum fuer Klinische Zusammenarbeit Wuerzburg (N-320)

  • Christina Lillesaar

Deutsche Forschungsgemeinschaft (ID 424778381)

  • Robert Blum

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All effort was taken to minimize the number of animals used and their suffering.Lab-Wue1: All experiments with C57BL/6J wildtype mice were in accordance with the Guidelines set by the European Union and approved by our institutional Animal Care, the Utilization Committee and the Regierung von Unterfranken, Würzburg, Germany (License number: 55.2-2531.01-95/13). C57BL/6J wildtype mice were bred in the animal facility of the Institute of Clinical Neurobiology, University Hospital of Würzburg, Germany.Lab Mue: Male All animal experiments with male C57Bl/6J mice (Charles River, Sulzfeld, Germany) were carried out in accordance with European regulations on animal experimentation and protocols were approved by the local authorities (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen).Lab-Inns1: Experiments were performed in adult, male C57Bl/6NCrl mice (Charles River, Sulzfeld, Germany). They were bred in the Department of Pharmacology at the Medical University Innsbruck, Austria. All procedures involving animals and animal care were conducted in accordance with international laws and policies (Directive 2010/63/EU of the European parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 2011) and were approved by the Austrian Ministry of Science.Lab-Inns2: Male 129S1/SvImJ (S1) mice (Charles River, Sulzfeld, Germany) were used for experimentation. The Austrian Animal Experimentation Ethics Board (Bundesministerium für Wissenschaft Forschung und Wirtschaft, Kommission für Tierversuchsangelegenheiten) approved all experimental procedures.

Reviewing Editor

  1. Scott E Fraser, University of Southern California, United States

Publication history

  1. Received: June 15, 2020
  2. Accepted: October 16, 2020
  3. Accepted Manuscript published: October 19, 2020 (version 1)

Copyright

? 2020, Segebarth et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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