QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy
Glyn Nelson, Ulrike Boehm, Steve Bagley, Peter Bajcsy, Johanna Bischof, Claire M Brown, Aurelien Dauphin, Ian M Dobbie, John E Eriksson, Orestis Faklaris, Julia Fernandez-Rodriguez, Alexia Ferrand, Laurent Gelman, Ali Gheisari, Hella Hartmann, Christian Kukat, Alex Laude, Miso Mitkovski, Sebastian Munck, Alison J North, Tobias M Rasse, Ute Resch-Genger, Lucas C Schuetz, Arne Seitz, Caterina Strambio-De-Castillia, Jason R Swedlow, Ioannis Alexopoulos, Karin Aumayr, Sergiy Avilov, Gert-Jan Bakker, Rodrigo R Bammann, Andrea Bassi, Hannes Beckert, Sebastian Beer, Yury Belyaev, Jakob Bierwagen, Konstantin A Birngruber, Manel Bosch, Juergen Breitlow, Lisa A Cameron, Joe Chalfoun, James J Chambers, Chieh-Li Chen, Eduardo Conde-Sousa, Alexander D Corbett, Fabrice P Cordelieres, Elaine Del Nery, Ralf Dietzel, Frank Eismann, Elnaz Fazeli, Andreas Felscher, Hans Fried, Nathalie Gaudreault, Wah Ing Goh, Thomas Guilbert, Roland Hadleigh, Peter Hemmerich, Gerhard A Holst, Michelle S Itano, Claudia B Jaffe, Helena K Jambor, Stuart C Jarvis, Antje Keppler, David Kirchenbuechler, Marcel Kirchner, Norio Kobayashi, Gabriel Krens, Susanne Kunis, Judith Lacoste, Marco Marcello, Gabriel G Martins, Daniel J Metcalf, Claire A Mitchell, Joshua Moore, Tobias Mueller, Michael S Nelson, Stephen Ogg, Shuichi Onami, Alexandra L Palmer, Perrine Paul-Gilloteaux, Jaime A Pimentel, Laure Plantard, Santosh Podder, Elton Rexhepaj, Arnaud Royon, Markku A Saari, Damien Schapman, Vincent Schoonderwoert, Britta Schroth-Diez, Stanley Schwartz, Michael Shaw, Martin Spitaler, Martin T Stoeckl, Damir Sudar, Jeremie Teillon, Stefan Terjung, Roland Thuenauer, Christian D Wilms, Graham D Wright, Roland Nitschke
QQUAREP-LiMi: A community-driven initiative toestablish guidelines for quality assessment andreproducibility for instruments and images in lightmicroscopy (cid:18)
Glyn Nelson , (cid:18) Ulrike Boehm , (cid:18) Steve Bagley , (cid:18) Peter Bajcsy , (cid:18) Johanna Bischof , (cid:18) Claire M Brown , Aur ´elien Dauphin , (cid:18) Ian M Dobbie , (cid:18) John E Eriksson , (cid:18) Orestis Faklaris , (cid:18) Julia Fernandez-Rodriguez , (cid:18) AlexiaFerrand , Laurent Gelman , Ali Gheisari , (cid:18) Hella Hartmann , (cid:18) ChristianKukat , (cid:18) Alex Laude , (cid:18) Miso Mitkovski , Sebastian Munck , (cid:18) Alison JNorth , (cid:18) Tobias M Rasse , (cid:18) Ute Resch-Genger , Lucas C Schuetz , (cid:18) ArneSeitz , (cid:18) Caterina Strambio-De-Castillia , (cid:18) Jason R Swedlow , (cid:18) IoannisAlexopoulos , Karin Aumayr , (cid:18) Sergiy Avilov , (cid:18) Gert-Jan Bakker , (cid:18) Rodrigo RBammann , (cid:18) Andrea Bassi , Hannes Beckert , Sebastian Beer , (cid:18) Yury Belyaev ,Jakob Bierwagen , Konstantin A Birngruber , (cid:18) Manel Bosch , Juergen Breitlow , (cid:18) Lisa A Cameron , Joe Chalfoun , (cid:18) James J Chambers , (cid:18) Chieh-Li Chen , (cid:18) Eduardo Conde-Sousa , (cid:18) Alexander D Corbett , (cid:18) Fabrice P Cordelieres , (cid:18) Elaine Del Nery , Ralf Dietzel , Frank Eismann , (cid:18) Elnaz Fazeli , Andreas Felscher , (cid:18) Hans Fried , Nathalie Gaudreault , (cid:18) Wah Ing Goh , (cid:18) Thomas Guilbert , RolandHadleigh , Peter Hemmerich , Gerhard A Holst , (cid:18) Michelle S Itano , Claudia BJaffe , (cid:18) Helena K Jambor , Stuart C Jarvis , Antje Keppler , DavidKirchenbuechler , Marcel Kirchner , (cid:18) Norio Kobayashi , (cid:18) Gabriel Krens , (cid:18) Susanne Kunis , Judith Lacoste , (cid:18) Marco Marcello , (cid:18) Gabriel G Martins , Daniel JMetcalf , (cid:18) Claire A Mitchell , (cid:18) Joshua Moore , (cid:18) Tobias Mueller , (cid:18) Michael SNelson , Stephen Ogg , (cid:18) Shuichi Onami , Alexandra L Palmer , (cid:18) PerrinePaul-Gilloteaux , (cid:18) Jaime A Pimentel , (cid:18) Laure Plantard , (cid:18) Santosh Podder ,Elton Rexhepaj , Arnaud Royon , Markku A Saari , (cid:18) Damien Schapman , VincentSchoonderwoert , (cid:18) Britta Schroth-Diez , Stanley Schwartz , (cid:18) Michael Shaw , (cid:18) Martin Spitaler , (cid:18) Martin T Stoeckl , (cid:18) Damir Sudar , Jeremie Teillon , (cid:18) StefanTerjung , (cid:18) Roland Thuenauer , (cid:18) Christian D Wilms , (cid:18) Graham D Wright , and (cid:18) Roland Nitschke Bioimaging Unit, Newcastle University, Newcastle upon Tyne, NE4 5PL, UK Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA Visualisation, Irradiation & Analysis, Cancer Research UK Manchester Institute, Alderley Park, Macclesfield, UK National Institute of Standards and Technology, Gaithersburg, MD 20899, USA Euro-BioImaging, Heidelberg, 69117, Germany Advanced BioImaging Facility (ABIF), McGill University, Montreal, Quebec, H3G 0B1, Canada Unit ´e G ´en ´etique et Biologie du D ´eveloppement U934, PICT-IBiSA, Institut Curie/Inserm/CNRS/PSL ResearchUniversity, Paris, 75005, France Department of Biochemistry, University of Oxford, Oxford, Oxon, OX1 3QU, UK Turku Bioscience Centre, Euro-Bioimaging ERIC, Turku, 20520, Finland Biocampus, CNRS UAR 3426, Montpellier 34293, France Centre for Cellular Imaging, Sahlgrenska Academy, University of Gothenburg, Gothenburg, 40530, Sweden Imaging Core Facility, Biozentrum, University of Basel, Basel, 4056, Switzerland Friedrich Miescher Institute for Biomedical Research, Basel, 4058, Switzerland Light Microscopy Facility, CMCB Technology Platform, TU Dresden, Dresden, 01307, Germany FACS & Imaging Core Facility, Max Planck Institute for Biology of Ageing, Cologne, 50931, Germany Light Microscopy Facility, Max Planck Institute of Experimental Medicine, Goettingen, 37075, Germany VIB BioImaging Core & VIB-KU Leuven Center for Brain and Disease Research & KU Leuven Department forNeuroscience, Leuven, Flanders, 3000, Belgium The Rockefeller University, New York, NY 10065, USA Scientific Service Group Microscopy, Max Planck Institute for Heart and Lung Research, Bad Nauheim, 61231,Germany Division Biophotonics, Federal Institute for Materials Research and Testing, Berlin, 12489, Germany European Molecular Biology Laboratory, Advanced Light Microscopy Facility, Heidelberg, 69117, Germany Faculty of Life Sciences, Ecole Polytechnique F ´ed ´erale de Lausanne, Lausanne, Vaud, 1015, Switzerland Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA Divisions of Computational Biology and Gene Regulation and Expression, School of Life Sciences, University ofDundee, Dundee, UK General Instrumentation - Light Microscopy Facility, Radboud University, Faculty of Science, Nijmegen, 6525,Netherlands BioOptics Facility, IMP - Research Institute of Molecular Pathology, Vienna, 1030, Austria Max Planck Institute of Immunobiology and Epigenetics, Freiburg, 79108, Germany Department of Cell Biology (route 283), Radboud Institute for Molecular Life Sciences, Nijmegen, 6525GA,Netherlands Scientifica Ltd, Uckfield, East Sussex, TN22 1QQ, UK Dipartimento di Fisica, Politecnico di Milano, Milan, 20133, Italy Microscopy Core Facility, Universit ¨at Bonn, Medizinische Fakult ¨at, Bonn, 53127, Germany Hamamatsu Photonics GmbH, Herrsching, 82211, Germany Microscopy Imaging Center, University of Bern, Bern, 3012, Switzerland AHF analysentechnik AG, Tuebingen, 72074, Germany TOPTICA Photonics AG, Graefelfing, 82166, Germany Fac. Biology, Prevosti’s building, Universitat de Barcelona, Barcelona, Spain PicoQuant, Berlin, 12489, Germany Light Microscopy Core Facility; Biology, Duke Univeristy, Durham, NC 27708, USA Institute for Applied Life Sciences, University of Massachusetts, Amherst, MA 01003, USA Pathware, Seattle, WA 98121, USA i3S - Instituto de Investigac¸ ˜ao e Inovac¸ ˜ao em Sa ´ude, Universidade do Porto, 4169-007 Porto, Portugal INEB - Instituto de Engenharia Biom ´edica, Universidade do Porto, 4169-007 Porto, Portugal Department of Physics and Astronomy, University of Exeter, Exeter, EX4 4QL, UK Bordeaux Imaging Center, Bordeaux, Nouvelle Aquitaine, 33077, France BioPhenics High-Content Screening Laboratory (PICT-IBiSA), Translational Research Department, Institut Curie -PSL Research University, Paris, 75248, France Omicron-Laserage Laserprodukte GmbH, Rodgau, 63110, Germany Carl Zeiss Microscopy GmbH, Jena, 07745, Germany University of Turku, Turku, 20520, Finland Coherent Lasersysems GmbH & Co.KG, Luebeck, 23569, Germany Light Microscope Facility, German Center for Neurodegenerative Diseases (DZNE), Bonn, 53127, Germany Allen Institute for Cell Science, Seattle, WA 98109, USA A*STAR Microscopy Platform, Research Support Centre, Agency for Science, Technology and Research,Singapore, 138648, Singapore Institut Cochin, INSERM (U1016), CNRS (UMR 8104), Universit ´e de Paris (UMR-S1016), Paris, 75014, France Core Facility Imaging, Leibniz Institute on Aging, Jena, 07745, Germany Research & Science, PCO AG, Kelheim, 93309, Germany Neuroscience Microscopy Core, University of North Carolina, Chapel Hill, NC 27599-7250, USA Lumencor, Inc., Beaverton, OR 97006, USA Mildred-Scheel Nachwuchszentrum, Universit ¨atsklinikum Carl Gustav Carus, TU Dresden, Dresden, 01307,Germany Prior Scientific Instruments Limited, Cambridge, Cambridgeshire, CB21 5ET, UK EMBL Heidelberg, Global BioImaging, Heidelberg, 69117, Germany Northwestern, Chicago, IL 60611, USA RIKEN, Wako, Saitama, 351-0198, Japan Bioimaging Facility, Institute of Science and Technology Austria, Klosterneuburg, 3400, Austria Biology/Chemistry, University Osnabrueck, Osnabrueck, 49080, Germany MIA Cellavie Inc., Montreal, Quebec, H1K 4G66, Canada Inst. of Systems, Molecular & Integrative Biology, University of Liverpool, Liverpool, Merseyside, L697ZB, UK Instituto Gulbenkian de Ciencia & Faculdade de Ciencias, Univ. Lisboa., Oeiras, 2780-156, Portugal Warwick Medical School, University of Warwick, Coventry, West Midlands, CV4 7AL, UK School of Life Science, University of Dundee, Walluf, 65396, Germany Gregor Mendel Institute of Molecular Plant Biology (GMI), Vienna, 1030, Austria City of Hope, Duarte, CA 91010, USA Medical Microbiology & Immunology, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada RIKEN Center for Biosystems Dynamics Research, Kobe, Hyogo, 650-0047, Japan Advanced Light Microscopy, The Francis Crick Institute, London, NW1 1AT, UK Universit ´e de Nantes, CHU Nantes, Inserm, CNRS, SFR Sant ´e, Inserm UMS 016, CNRS UMS 3556, F-44000Nantes, France Instituto de Biotecnolog´ıa, Universidad Nacional Aut ´onoma de M ´exico, Cuernavaca, Morelos, 62210, Mexico Microscopy Facility, Biology, Indian Institute of Science Education and Research Pune, Pune, 411008, India Sanofi Aventis, Chilly-Mazarin, Essone, 91380, France Argolight, Pessac, 33600, France Turku Bioscience Centre, University of Turku and ˚ Abo Akademi University, Turku, 21520, Finland Normandie univ, UNIROUEN, INSERM, PRIMACEN, 76000 Rouen, France Scientific Volume Imaging bv, Hilversum, Noord-Holland, 1213VB, Netherlands Light Microscopy Facility, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, 01307, Germany Nikon Instruments Inc. ISO Consultant, Melville, NY 11747, USA National Physical Laboratory, Teddington, Middlesex, TW11 0LW, UK Imaging Facility, Max Planck Institute of Biochemistry, Martinsried, Munich, 82152, Germany Bioimaging Center, University of Konstanz, Konstanz, 78464, Germany Quantitative Imaging Systems, Portland, OR 97209, USA Bordeaux Imaging Center, Universit ´e de Bordeaux, Bordeaux, Gironde, 33076, France Technology Platform Microscopy and Image Analysis, Heinrich Pette Institute, Leibniz Institute for ExperimentalVirology, Hamburg, 20251, Germany Life Imaging Center and BIOSS Centre for Biological Signaling Studies, Albert-Ludwigs-University Freiburg,Freiburg, 79104, Germany * these authors contributed equally to this work x these authors are active members of QUAREP-LiMi’s working group 8 and contributed significantly to therealization of this paper + Corresponding author. Email: [email protected] . 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BSTRACT
A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now asophisticated, quantitative device that is an integral part of both physical and life science research. Nowadays, microscopesare found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientificphenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriateknowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated bythe well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducingscientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat anotherscientist’s experiments, while more than half have even failed to reproduce their own experiments . One factor behind thereproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods causedby a lack of awareness and/or a lack of knowledge of the applied technique . Whereas quality control procedures for somemethods used in biomedical research, such as genomics (e.g., DNA sequencing, RNA-seq) or cytometry, have been introduced(e.g. ENCODE ), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibrationstandards and protocols have been published, there is a lack of awareness and agreement on common standards andguidelines for quality assessment and reproducibility .In April 2020, the QUality Assessment and REProducibility for Instruments and Images in Light Microscopy (QUAREP-LiMi)initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interestin achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) inlight microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines,metadata models, and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientificresearch.This White Paper 1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMiinitiative; 2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholdersincluding, researchers, imaging scientists , bioimage analysts, bioimage informatics developers, corporate partners, fundingagencies, standards organizations, scientific publishers, and observers of such; 3) outlines the current actions of the QUAREP-LiMi initiative, and 4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposedguidelines to manage QC.To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of lightmicroscope image data by introducing broadly accepted standard practices and accurately captured image data metrics. Preface
The QUality Assessment and REProducibility for Instruments and Images in Light Microscopy (QUAREP-LiMi) initiative a aims at convening the light microscopy community with the explicit purpose of reaching a broad consensus concerning QualityControl and Quality Assessment guidelines for optical microscopy to be adopted worldwide. For the purposes of this discussion,by “light microscopy community,” we refer to everyone working directly or indirectly with light microscopes and image data,independent of the specific microscope design or configuration. Although we aim to satisfy the entire community’s requirementsand views, we cannot claim sufficient diversity or coverage of the community for complete representation. Rather, this WhitePaper is the first of a series that will report our ongoing progress towards achieving the stated goals of QUAREP-LiMi. Whilethe work of QUAREP-LiMi aims at developing recommendations and guidelines that can be easily extended across disciplines(both physical and life sciences), for the sake of simplicity, the discussion is currently restricted to applications and examplesdrawn mainly from biology. Although our current efforts focus on establishing guidelines for widefield and confocal opticalmicroscopes, we are keen to extend the breadth of our work subsequently to cover other light-microscopy-based imagingmodalities. Background
Current Situation
Since their introduction in the early 17th Century, microscopes have transitioned from basic, qualitative image-collecting toolsto sophisticated instruments capable of automatically acquiring information-rich images that are further processed via advancedimage processing and analysis steps to extract quantitative information about the underlying science. The robustness of theconclusions that we make from these observations will depend upon the reproducibility of the samples and the microscopesystem used to image them. Importantly, each instrument’s technical characteristics need to be fully understood and documentedto permit valid interpretation of imaging data. To enable the reliable and reproducible extraction of quantitative information, a https://quarep.org/ igure 1. Acquiring imaging data that is both quantifiable and reproducible involves a myriad of factors, few ofwhich are acknowledged or accurately recorded.
Intimate knowledge of the composition and performance of a system isessential for reproducibility. However, performance measurements may be tricky, and require specific protocols, tools, samples,training, and data analysis methods. In order to help microscope users to assess and judge the performance of their systemsproperly, the community must agree on and publish guidelines and benchmarks.microscopes, including advanced widefield and confocal instruments, must therefore be well described, maintained, calibrated,and in essence ‘quality controlled’ (Figure 1).Unfortunately, the requirement for robust microscope and image quality assessment is not commonly recognized amongst thescientific community, leading to the infrequent application of appropriate QC procedures. This is due to several barriers:1. The lack of widely adopted community-wide guidelines and standards for light microscopy documentation and QC;2. The limited willingness of the community’s stakeholders (researchers, funders, and scientific publishers) to enforceexisting guidelines and standards;3. Insufficient training of microscope users on the complexity of performing quantitative imaging and on guidelines andstandards for quality assessment and reproducibility.As a result, rigor and reproducibility are limited, the reliability of quantitative analysis is severely impacted, and the confidencein published data becomes eroded.Encouragingly, the awareness of the importance of QC and reproducibility in light microscopy has gained traction in recent years,both within the scientific community and amongst funders (e.g., National Institutes of Health (NIH), European Research Council(ERC)) as well as within newly launched bioimaging networks (e.g., Max Planck BioImaging Core Unit Network b ). Moreactive steps have been taken within several microscopy initiatives such as Global Bioimaging (GBI c ), Euro-BioImaging ERIC(European Research Infrastructure Consortium) d , the Royal Microscopical Society (RMS e ), German BioImaging-Gesellschaftfür Mikroskopie und Bildanalyse (GerBI-GMB f ), BioImaging North America (BINA g ) and the RT-MFM technological network(Microscopie photonique de Fluorescence Multidimensionnelle h ). GBI has published an overview of the current landscapefor quality assurance and data management in imaging facilities , including recommendations for QC. This document alsohighlights multiple aspects concerning image data standardization, management, and publication, such as the definition of imageand microscopy metadata guidelines and data models and the need to provide open access to all raw data for acceptedmanuscripts (e.g., Image Data Resource and BioImage Archive) , which are being addressed both within QUAREP-LiMi(see Working Group 7 – Metadata) and by others within the imaging community. The Euro-Bioimaging ERIC includesthe independent assessment of QC measures and implementation in the ongoing evaluation of existing Nodes and during theapplication process for new imaging Node candidates. Finally, BINA, the RMS, GerBI-GMB, and the RT-MFM are all actively b c d e f g h http://rtmfm.cnrs.fr/ ngaged in tackling QC and reproducibility issues via dedicated working groups (“QC and Data Management (QC-DM i );“QC Focussed Interest Group” j ; “Quality Assessment for Instruments & Facilities” k ; and “Metrological Measurements onMicroscopy,” respectively). Current Approaches
Despite the importance of individual local efforts, they prove insufficient to overcome the global challenges associated withQC in light microscopy. In the following section, we highlight a few of these approaches and discuss why they are unable tocompletely overcome existing hindrances individually.
Quality Control procedures adopted by individual core facilities and laboratories
To tackle common QC issues, many core facilities and laboratories regularly perform maintenance and various QC tests oftheir instruments. However, the nature and frequency of the performed tests vary greatly, depending on the priorities set byresearchers, imaging facility staff, and their institution. A survey initiated by the European Light Microscopy Initiative (ELMI)in 2019 l highlighted that numerous core facilities and labs already perform QC, but a considerable percentage does not at all(Figure 2a). Likewise, there was wide variation in the respondents’ choice of tools, making any comparison and reproducibilityof QC results between equipment difficult (Figure 2b). Guidelines by the International Organization for Standardization (ISO)
The International Organisation for Standardization (ISO m ) has created standards for brightfield microscopy and, morerecently, for confocal microscopy . These ISO standards provide researchers with directions as to what should be measuredand tested. Nevertheless, there is little information describing how key measurements should be made within these documents,using which samples and tools, and with what frequency . Tools and protocols by the community for the community
Several individual groups have published methods and software tools to streamline and automate microscope QC procedures(e.g. and recently reviewed in ). In addition, several open-source software tools that provide different degrees ofautomation for different microscopy calibration tasks have been developed and made available both as ImageJ-based macros andplugins (e.g., NoiSee , MetroloJ , ConfocalCheck , AutoQC , PSFj and MIPs for PSFs , SIMcheck ) and standaloneweb applications (e.g., PyCalibrate n ). Finally, international endeavors involving the global community were carried out andpublished by the Association of Biomolecular Resource Facilities (ABRF) . These efforts provide both valuable results andmetrics that can be saved locally and archived individually. However, these undertakings were affected by significant variationsbetween individual groups. Moreover, they are neither comprehensive nor address standardization of metadata capture, andthey are not fully aligned with the recommendations put forth by standards organizations such as the ISO. Regulations and guidelines imposed by third parties
Besides the ISO, funding agencies, scientific publishers, and community organizations (e.g., GerBI-GMB, RMS, BINA,RT-MFM) often furnish QC guidelines for light microscopy. However, these guidelines are not exhaustive, are often issued inisolation, and are not accepted by the principal constituents of the imaging community (including commercial microscopemanufacturers and the broader scientific community).All of these approaches share a similar set of limitations: 1) they are currently adopted voluntarily and are therefore un-enforceable; 2) they are often targeted at highly trained imaging facility staff and are often not accessible to less expert,non-facility microscope users, and custodians in individual laboratories; 3) they are limited in scope and therefore do notguarantee proper QC and reproducibility, and 4) they are not standardized and therefore show significant variability. The reasonsfor this are several-fold. Firstly, there is a lack of agreement regarding the recommended standard samples, tools, protocols,and metrics to be measured. Hence, there is a pressing need for the community to agree on what should be measured for eachhardware component (e.g., laser, camera, or objective lens) and calibration procedure (i.e., optical, intensity, and mechanicalcalibration), which tools and samples should be used, and the frequency of QC measurements for different metrics. Secondly, acommonly cited reason for minimizing or avoiding microscope QC is the lack of appropriate time and resources (i.e., personnel,machine time, and required hardware) afforded to microscope custodians to perform the appropriate tests, the downstreamanalysis, and the compilation of the results across time accurately and systematically. Finally, most protocols/methods currentlybeing performed are marred by high variability of the measured values, almost entirely due to the lack of automation. i j k l https://lic-machform.vm.uni-freiburg.de/view.php?id=59721 m n Frequency of Quality Checks at Microscopy Facilities. (b)
Tools used for Performance Evaluation in Light Microscopy (ordered according to popularity).
Figure 2.
Frequency and type of tests performed in core facilities.
Prior to a session on Quality Control organized duringthe Microscopy Facility Day at the 2019 ELMI meeting, light microscopy core facility representatives around the globe wereasked to complete a survey about the type and the frequency of tests performed in their facility. The link to the survey wasopened in June 2019, two weeks before the meeting, and sent to all registered participants. It was also advertized multiple timeson various international microscopy forums. Reminders were also sent to participants after the meeting and responses weresubsequently collected until February 2020. The histograms in panels (a) and (b) summarize the responses from almost 200facilities in a simplified manner. Panel (a) displays how often different quality checks are performed; the x axis represents inpercentage the respective frequency categories, namely regularly, on demand, and never. Panel (b) highlights which tools areused for performance evaluation of light microscopes; the x axis represents the percentage of respondents using the indicatedQC tool. roposed Community-Driven Approach
Following a discussion at the 2019 conference of the European Light Microscopy Initiative (ELMI 2019), members of theGerBI-GMB and RT-MFM networks launched a shared strategy to build a community consensus on QC measurements. Thisinitial initiative rapidly integrated with similar efforts being conducted by the BINA QC-DM working group o and the RMSQC focussed interest group p . Shortly after, the publication of the Confocal ISO 21073 provided the scientific communitywith an agreed minimal set of tests that should be performed to assess the performance of confocal microscopes. Based uponthis publication, a methodology manual was drafted to describe how the ISO-recommended QC metrics could be obtained inpractice and presented to participants representing academia, industry, and governmental standardization bodies at a meetingheld on April 28th, 2020. This led to the formal establishment of QUAREP-LiMi q (coordinated by R. Nitschke, Gerbi-GMB).QUAREP-LiMi is a grass-root global community that is open to individuals from academia, industry, government, fundingagencies, and scientific journals from around the world with interest in improving QC in light microscopy. As a testament to thetimeliness of this strategy, QUAREP-LiMi quickly grew from 49 initial participants to 184 (updated 05/01/2021) individuals (atthe time of writing) from 20 countries (Figure 3). Compared to earlier approaches (see section “Current Approaches”), theQUAREP-LiMi initiative is specifically designed to work in a completely transparent and open manner to foster ground-upparticipation from around the globe and ownership by all members of the community. By taking into account all existingapproaches and recommendations, the specific goal is to produce a consensus around shared, binding QA and QC guidelinesand specifications for the scientific community and corporate partners. Furthermore, QUAREP-LiMi will work with bothjournals and funders to encourage stakeholders to adopt and enforce these standards. Figure 3.
Summary of current QUAREP-LIMi participants according to their origin and affiliation (updated: January5, 2021) . o p q https://quarep.org/ ey Beneficiaries The deliverables from QUAREP-LiMi will benefit several groups related to light microscopy, from image data acquisition allthe way to image data processing, presentation, sharing, and reuse.1.
Research scientists and imaging scientists will take advantage of the harmonization and simplification of the QCprocedures, facilitation of QC capture and storage, and clear interpretation of QC results to better understand how theperformance of their microscope impacts the interpretation of scientific results.2.
Scientific publishers and the general public will profit from an overall enhanced trust in the value and reproducibilityof scientific publications, resulting from the publication of full descriptions (i.e., Material and Methods sections andmicroscopy metadata) of the technical make-up of microscopes and of performance metrics to accompany raw imagedata.3.
Funding bodies will benefit from the planned improvement of QC practices that will undoubtedly improve image datareliability, reproducibility, and openness, increasing overall the value and quality of scientific output and opening the wayto truly FAIR data . Besides, the improved likelihood of data reuse towards novel discoveries will significantly impacttaxpayer funds’ efficient use.4. Core imaging facilities and their users, as well as non-facility microscope custodians and users, will profit fromheightened confidence in the accuracy of their image data and the suitability of performed QC measurements to answertheir scientific questions. Improving, prioritizing, and streamlining the various required QC procedures will ease the timeburden, facilitate the comparison of experimental data and improve the communication between imaging facility staffand commercial manufacturers based on common vocabulary, tools, and protocols. As an added advantage, communitystandard procedures will allow users to submit standardized performance metrics with their published imaging data,which will greatly facilitate the interpretation, reproducibility, and re-use of the results.5. Commercial microscope and system component manufacturers will be able to take advantage of the availability oftime-stamped and standardized microscope metrics to identify common microscope performance and quality issues,such as identifying faulty parts. They will also be able to utilize the metrics for future developments and the continuedimprovement of hard- and software products.
Initiated Steps and Dissemination
Although initiated around the Confocal Microscope ISO 21073 standard , the scope of QUAREP-LiMi has since increased. Itis now devoted to establishing a comprehensive set of shared QC guidelines, tools for their capture, and microscopy metadataspecifications for their storage and automated reporting.Direct and open engagement of the global imaging community, and public and effective dissemination of QUAREP-LiMiadvances, are essential to foster consensus-building and global acceptance of QUAREP-LiMi proposals. Consistent with thisgoal and its foundational principles, QUAREP-LiMi actively seeks participation from any interested party to join the group andwork towards establishing a global QC consensus. To this aim, regular updates on the progress achieved by individual workinggroups will be disseminated using various social platforms and the QUAREP-LiMi website for all interested parties to provideinput. Organizational Structure and Immediate Goals
Having established the general goals and founding principles of the group, members of QUAREP-LiMi agreed upon a set ofessential topics to address and established the following organizational structureon July 9th, 2020:1. The group aims to achieve specific deliverables and established that work will be conducted by individual workinggroups, each led by an elected Chair and Vice-Chair.2. All interested parties are welcome to participate, either as observing members or as active participants within one ormore working groups of their choice.3. All participants, regardless of whether they are observing or active participants, will be allowed to provide feedback onthe deliverables produced by individual working groups.4. The number of working groups will be extended as needed to ensure coverage of all aspects of microscopy QC andsatisfy all key beneficiaries’ desiderata.More specifically, the following working groups were established, and when appropriate, will produce a robust, easy-to-useprotocol based upon standardized samples or tools:
G1 Illumination Power
Comparison of fluorescence intensities between images requires measurements of the illumination power and stability of theexcitation light source. WG1 aims at establishing a recommended protocol for measuring the stability of a light source duringboth short- and long-term image acquisition sessions using calibrated external power sensors. This initial aim will be extendedlater to measure the absolute flux of light through the illumination path and irradiance of the sample. The initial protocol willbe designed around lasers on confocal microscopy platforms (raster scanning and spinning disks). It will be later modifiedtowards other microscopy techniques (widefield, TIRF, light-sheet, super-resolution).
WG2 Detection System Performance
WG2 focuses on the detection system, comprising the detection path and its detector(s), and how it measures the signal fromthe sample. Members of WG2 aim to standardize the characterization of the detection system performance and create standardprocedures for monitoring it over time, thereby revealing performance issues that could affect data reproducibility. Therefore,WG2 will define universal, externally measurable parameters applicable to any type of detector (e.g., photons, linearity, noise),together with measurement tools and protocols for measuring these parameters from common detector types. These universalparameters will be specified according to each distinct type of detector’s internal parameters, which have already been definedby the community. They will enable the evaluation and comparison of different detection systems, thus pinpointing the mostsuitable technology for given applications.
WG3 Uniformity of Illumination Field - Flatness
Illumination field uniformity is critical for quantitative imaging when comparing fluorescence intensities across a field-of-view(FOV) or a large tile of images capturing an entire sample. If the illumination is not constant over a large area, the fluorescenceintensities will not represent the inherent fluorescence but rather the location within the image. Thus, WG3 aims at defining aset of universal protocols to assess the uniformity of illumination (i.e., “flatness” of field) over the FOV of any photon-basedimaging system and allow for correction of any non-uniformity. These protocols will identify the necessary tools, the proceduresrequired to perform the measurements, and the analysis methods required for their interpretation. WG3 will also define criteriaregarding the cut-off for acceptability and the need for correction. A database will be created with ideal images of uniformfields-of-view from different microscope modalities and settings to be used as a reference by the community and validate theprotocol and criteria.
WG4 System Chromatic Aberration and Co-Registration
Chromatic aberration refers to possible artifacts caused by the wavelength dependency of an imaging system’s optical properties,with the result that two colors arising from the same physical location within the sample appear separated in the image.Such artifacts result from the optical design of the system (e.g., well-corrected versus poorly corrected objective lenses), themanufacturing tolerances of the system components, and the alignment of the optical components. Co-registration accuracymore generally refers to the system’s ability to co-localize dyes of different wavelengths emitting from the same object within aparticular experimental set-up. This can be affected by both the experimental set-up and the system architecture. Working withinthe assumption that microscope users are ultimately interested in co-registration accuracy, WG4 aims to use sub-resolution andlarger multi-colored bead preparations to measure co-registration accuracy. Alternative tools for performing these measurementswill also be evaluated. WG4 will compare reproducibility across different laboratories to determine the best protocol.
WG5 Lateral and Axial Resolution
This WG focuses on the microscope lateral and axial resolution, which is essential for reporting size measurements of near-resolution limit objects or distances between them. Resolution is highly related to the objective quality but depends stronglyupon other parameters ranging from the sample preparation to the signal detection.The WG aims to define sample preparation, image acquisition, and data analysis protocols for testing resolution, first usingsub-resolution fluorescent bead preparations and second employing alternative pattern-based methods. Monitoring the resolution(Point Spread Function in the case of beads) over time will identify possible aberrations in the system. Pooling the data frommultiple laboratories within the WG will allow them to compare reproducibility for sample preparation, data acquisition, anddata analysis tools, thereby determining a robust, easy-to-use protocol to propose to the community.
WG6 Stage and Focus - Precision and Other
The mission of WG6 is to ensure the performance and QC of stage platforms and sample holders and the optomechanical focusof the optical system as it relates to X, Y, Z movement, stability, reproducibility, and repeatability. The goals are defining theterms typically used to address QC, providing standardization of the measurements and testing protocols, and establishingperformance benchmarking levels. Though initially applying these towards confocal light microscopy, the WG will endeavor toinclude details for standard incident light fluorescence microscopy and more advanced techniques such as super-resolution andlight-sheet microscopy.
G7 Microscopy Data Provenance and QC Metadata
For proper interpretation, microscopy images must be accompanied by both human-readable (i.e., Materials and Methodssections) and machine-readable (i.e., metadata) descriptions of all steps leading to image formation (i.e., ‘data provenance’metadata) as well as by QC metrics detailing the illumination, detection, chromatic, optical and mechanical performance ofthe microscope. Nevertheless, no universally accepted community guidelines exist defining what ‘data provenance’ and QCmetadata should be reported for distinct types of imaging data. Therefore, the metadata automatically recorded by differentcommercial microscopes can vary widely, posing a substantial challenge for microscope users to create a bona fide record oftheir work. To meet these challenges, the 4D Nucleome (4DN) Imaging Working Group and the BINA QC-DM WG r havedeveloped a tiered set of Microscopy Metadata guidelines and a suite of extensions of the OME Data Model that scale withexperimental complexity and requirements, and are specifically tailored at enhancing comparability and reproducibility inlight microscopy. WG7 aims to systematically evaluate the structure and semantics of the initial 4DN-BINA-OME extensionproposal and to launch a coordinated outreach strategy towards reaching a wide community consensus around the proposedmetadata specifications. WG8 White Paper
The remit of WG8 is to relay both short and long-term goals of QUAREP-LiMi by the publication of a set of White Papers tocommunicate and seek cooperation from the community. The principal aim of these White Papers is to promote QUAREP-LiMito 1) Prospective new members: to actively engage with the work of QUAREP-LiMi; 2) Imaging scientists and bioimageanalysts: to raise awareness of QC issues; 3) Group Leaders/Principal Investigators: to engage a critical mass of academicresearchers (top-down); 4) Research scientists (graduate students and postdoctoral researchers) with expertise in the specializedWG topics and imaging scientists: to influence the research group leaders (bottom-up); 5) Scientific publishers: to raisethe quality of methods reporting and rigor and reproducibility in publications; 6) Leads (CEO/directors) of companies andcommercial application specialists: to work alongside QUAREP-LiMi to facilitate ease of measurements and reporting, and 7)Prospective funders (funding agencies, private sponsors): to support the work of this initiative.
WG9 Overall Planning and Funding
The principal aim of WG9 is to coordinate and promote the activities of QUAREP-LiMi. Within this WG, there is representationfrom all other WGs in addition to key global, regional, and national microscopy communities. WG9 will also liaise directlywith corporate partners, scientific publishers, and funding bodies.WG9 will focus on the following activities: 1) Ensure that the output of QUAREP-LiMi achieves maximum impact withinthe imaging community by raising awareness of the need for QC across all stakeholders in light microscopy (via whitepaper, website, publications); 2) Seek to obtain support from our corporate partners (microscope manufacturers/technologycompanies); 3) Obtain funding and support from national bodies, scientific publishers and learned societies to help cover theactivities of QUAREP-LiMi (allow us to stage physical meetings, cover publication costs, help with organization and addimpact); 4) Keep stakeholders informed and share information through a regularly updated website and tools database (towardsinternal and external communication and impact), and 5) Coordinate QUAREP-LiMi WGs and future QUAREP-LiMi meetings(virtual and physical).
WG10 Image Quality
Good image quality (IQ) is essential for any subsequent image processing, analysis, and presentation steps. However, thenotion of IQ is very broad and encompasses concepts that might differ between various microscope types. The aims of WG10are 1) to define a set of basic IQ parameters (quantitative criteria, metadata, QC metrics) for light microscopy; 2) to weightthe significance of the individual parameters for different experimental techniques and microscope types; and 3) to facilitatethe assignment of a microscope- and experiment-specific QC rating to individual images. Ultimately, WG10 will work tosummarize the upshot of these steps in the form of easy-to-use workflows. The integration of IQ ratings as part of the metadataassociated with every imaging dataset is a long-term goal of this WG.
WG11 Microscopy Publication Standards
WG11 will work together with scientific publishers to promote the adoption of best practices in the reporting of metadata(for both image acquisition and analysis) throughout scientific journals and books. Only by ensuring all relevant constituents(researchers and imaging scientists submitting publications and designing research; editors, scientific publishers, and reviewersmonitoring and preparing publications; and funders, researchers, and educators evaluating and disseminating publications) areworking in concert can we raise the bar to ensure reproducibility in imaging experiments. WG11 will focus on the followingactivities: 1) inform scientific publishers of the standards and metadata put forward by the other QUAREP Working Groups;2) liaise with and encourage individual journals to modify their imaging guidelines to align with these recommendations; 3)work together with the scientific publishers to enforce high standards of imaging metadata reporting in all research works r ccepted for publication; 4) facilitate the involvement of technical reviewers with significant microscopy expertise duringthe review of papers that rely heavily on imaging techniques; 5) work together with publishers to promote and increase theappropriate acknowledgement and co-authorship of imaging scientists and core imaging facilities in publications; 6) encouragepublishers to compel authors to make raw imaging data available if, and when, required for validation of published research andto make reasonable suggestions regarding duration of storage of raw imaging data relevant to published results; 7) proposeminimum standards for figure quality, figure colour selection, scale bars, inserts, annotations and labelling, in order to render allmicroscopy figures easily interpretable by experts and non-experts alike. Future Steps and Perspectives
The ultimate goal of QUAREP-LiMi is to benefit everybody in the light microscopy community. Our future strategy can besubdivided into medium-term goals to be achieved within the next few months and long-term goals to be realized within thenext 1-2 years.
Medium-term goals
Growth and diversification of QUAREP-LiMi member body:
The vast majority of current QUAREP-LiMi members areimaging scientists representing academic labs, core imaging facilities, and standardization organizations. For the mission ofQUAREP-LiMi to be successful, it is imperative to achieve greater engagement with industry (currently 17 companies with25% of the total members), scientific publishers, funding agencies, and commercial microscope manufacturers. Moreover, ahigh priority is to achieve a better worldwide representation of the imaging community by including more members outsideNorth America and Europe.
Establishing a consensus of accepted guidelines within the WGs:
Each WG working towards a defined QC method and overallmicroscopy metadata specification will finalize a proposed solution and methodology for their topic. They will present this tothe entire QUAREP-LiMi community (see “Organizational Structure and Immediate Goals” section) for evaluation, includingsome beta-testing. This will result in revised versions of individual WGs’ proposals submitted for final approval by the imagingcommunity. The final approval of guidelines and methods will be a community decision and result in documented and openlyaccessible QC protocols. This kind of workflow is adopted and slightly modified from the proven workflow of the ISO.
Long-term goals
Dissemination of new guidelines to the scientific community and its stakeholders:
The QUAREP-LiMi guidelines will bepublished like those for RNAseq, proteomics, microarrays, etc. , and highlighted at national and international scientificmeetings. Furthermore, the inclusion of the guidelines in teaching materials and training courses (both for microscope users andfor imaging facility staff , as well as for commercial developers and corporate partners) will ensure their wide-spread adoptionamong microscope users and developers. The QUAREP-LiMi guidelines, initially developed for widefield and confocalmicroscopy, are intended to be adopted and extended to other imaging modalities, such as light-sheet and super-resolutionmicroscopy. Implementation of the new guidelines within the community:
By engaging the entire imaging community throughout thedevelopment of new guidelines and specifications, we strive to implement a standard procedure for end-users and to promotethe integration of the guidelines into commercial microscope hardware and software. As this process becomes easier andmore streamlined, the long-term aim is to enable the automatic measurement of these metrics. Thus, early engagement withcommercial manufacturers and other developers is critical to ensure simple approaches towards acquiring QC data.
Working with stakeholders to promote the implementation of new guidelines:
A straightforward solution to encourage the uptakeof minimal QC metrics would be for scientific publishers to adopt these standards as part of their standard requirements toaccept material for publication. Ideally, access to raw data should also be provided. Such initiatives are gaining ground andbeing backed at the national level by funding bodies. Databases to store the QC data for each microscope should enable creatingsimple reports to accompany published experimental results, demonstrating the system’s real-time performance across thedata collection period. In other fields (genomics, transcriptomics, proteomics, etc.), public data repositories have played a keyrole in implementing community-proposed standards and accelerating their adoption. Data repositories and journals workedtogether as de facto enforcers of a standard. Journals required data related to peer-reviewed manuscripts to be submitted torepositories. In contrast, repositories themselves enforce standards either at the time of submission or by converting submitteddata to a standardized format for publication and download. With the establishment of several bioimage data publicationsystems , there is now an opportunity within the field of light microscopy to use a similar approach based on thesesuccesses. odification of the existing ISO and establishing of new ISO standards based on guidelines developed by QUAREP-LiMi:
The final formalization of the QUAREP-LiMi guidelines will be achieved by their inclusion in new editions of the respectiveISOs . Whilst we expect that microscope QC will be a constantly evolving area, as new technologies become mainstream,the establishment of fixed versioning of the current guidelines for widefield and confocal systems will provide the communitywith a strong baseline for further developments of ISOs. It will cover the vast majority of current microscopy-based research.
Conclusion
The international nature, size, and breadth of QUAREP-LiMi is critical for its mission, which will only succeed withsufficient buy-in from all stakeholders. The first step will be to reach a consensus between microscope and system componentmanufacturers, users, and microscope custodians regarding precisely what needs to be measured, how, and at what frequency,taking into account the experiment being performed and the downstream image analysis strategy. Next, a set of common,practical tools to accomplish these measurements must be developed and provided to the entire community. The microscopemanufacturers can provide some of these as internal QC tools that align with the QUAREP-LiMi guidelines, thereby facilitatingrapid, simple measurements by all microscope users and custodians. Such tools would help the companies ensure theirinstrumentation’s consistent performance, facilitate the more rapid diagnosis of problems, and permit imaging scientists toperform necessary checks and alignments that must currently be performed by dedicated service engineers. Commercialmicroscope manufacturers can also support this paradigm shift by raising awareness of the importance of imaging QC withtheir direct customers; thus, their involvement in the QUAREP-LiMi initiative and working groups is crucial.An equally critical factor in the agreed-upon guidelines’ global adoption will involve education and raising awareness throughpublications, workshops, and meeting presentations explicitly targeted at research and imaging scientists. Since a significantpart of many imaging scientists’ responsibilities already lies in maintaining instrumentation at optimal performance, they aretypically more familiar with the problems and challenges outlined above. Hence, they have tremendous potential to educateresearchers on the importance of imaging QC, the tools available and recommended, and to disseminate the QUAREP-LiMiguidelines to their facility users and researchers who have microscopes in their laboratories. Funding bodies and scientificpublishers could also encourage adopting these guidelines by requiring their implementation in all imaging-focused research.Scientific publishers can further educate their reviewers in imaging QC or bring in expert technical assessors to interrogatedata quality and reliability. Publishers and reviewers should also encourage the sharing of imaging data in public repositories.Finally, repository hosts could help enforcement by automating data quality validation and ensuring that the data is made widelyavailable to the broader scientific community.QC is costly and requires significant time and effort, but its lack undermines trust in the quality of data, equipment, scientificrigor, reproducibility, and data exchange. By providing a clear community-driven way forward and working closely withall stakeholders, QUAREP-LiMi has the potential to drive a culture change. This will benefit the entire community byfundamentally transforming image data quality and reproducibility.
Acknowledgments
We thank somersault18:24 BV (Leuven, Belgium) for help with Figure 1. E. C.-S. was supported by the project PPBI-POCI-01-0145-FEDER-022122, in the scope of Fundação para a Ciência e Tecnologia, Portugal (FCT) National Roadmap of ResearchInfrastructures.
Author contributions
U.B. and G.N. coordinated the QUAREP-LiMi White Paper working group (WG8) and the effort to write this manuscript. Allactive members of the WG8 contributed significantly to the realization of the manuscript (from conceptual development andwriting to the creation of the figures and glossary). R.N. coordinates the QUAREP-LiMi initiative.
Competing interests
R.R.B., R.H., D.J.M., C.D.W., S.B., J.B., K.A.B., J.B., R.D., F.E., A.F., W.I.G., G.A.H., C.B.J., S.C.J., J.L., E.R., A.R., V.S.,S.S., and D.S. have a financial interest as employees of companies producing, selling or distributing light microscopes orproducts related or used with light microscopy equipment. A.D.C. has a financial interest in PSFcheck and PyCalibrate.
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