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.
- Robert Blum
- Hans-Christian Pape
- Maren D Lange
- Rohini Gupta
- Manju Sasi
- Ramon O Tasan
- Nicolas Singewald
- Christina Lillesaar
- Robert Blum
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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.
- Scott E Fraser, University of Southern California, United States
- Received: June 15, 2020
- Accepted: October 16, 2020
- Accepted Manuscript published: October 19, 2020 (version 1)
? 2020, Segebarth et al.
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