Best practices and tools for reporting reproducible fluorescence microscopy methods

Montero-Llopis, Paula; Senft, Rebecca A.; Ross‐Elliott, Timothy J.; Stephansky, Ryan; Keeley, Daniel P.; Koshar, Preman; Marqués, Guillermo; Gao, Ya-sheng; Carlson, Benjamin R.; Pengo, Thomas; Sanders, Mark A.; Cameron, Lisa; Itano, Michelle S.

doi:10.1038/s41592-021-01156-w

Cited by 81 publications

(65 citation statements)

References 64 publications

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“…Reproducibility of fluorescence microscopy methods was previously identified as a major issue [41]. Quantitative fluorescence microscopy is inherently difficult to reproduce, due to the large number of factors involved.…”

Section: Discussionmentioning

confidence: 99%

Rapid single-molecule characterisation of nucleic-acid enzymes

Mueller

Fitschen

Shirbini

et al. 2022

Preprint

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The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lacking statistical power. We present a highly generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different nucleic-acid enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe a previously unknown activity of the UvrD helicase to remove proteins tightly bound to the ends of DNA.

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Section: Discussionmentioning

confidence: 99%

Rapid single-molecule characterisation of nucleic-acid enzymes

Mueller

Fitschen

Shirbini

et al. 2022

Preprint

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“…To coat the coverslip, we dipped the coverslip in PolyLys solution for 2 min, washed it with water, and dried it. The slides were visualized using a Zeiss 780 confocal microscope equipped with a 63×/1.4 Plan-Apochromat oil immersion objective ( 47 ). Images were processed by merging the two-color channels in FIJI, a distribution of imageJ2, without changing the pixel intensity ( 48 ).…”

Section: Methodsmentioning

confidence: 99%

Strain-Level Variation and Diverse Host Bacterial Responses in Episymbiotic Saccharibacteria

et al. 2022

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“…A recent exploration about the quality of published Method sections in scientific articles containing images obtained with advanced microscopes, found that the quality of reporting was poor, with some articles containing no information about how images were obtained, and many articles lacking important basic details (13). Nonetheless, there is ample evidence that the publication of full details about how each image was obtained is vital for rigor, reproducibility and maximal scientific and sharing value (14)(15)(16)(88)(89)(90). In this context, the 4DN-BINA-OME Microscopy Metadata specifications presented are intended to provide a major contribution towards the development of community-driven criteria for which information should be included in the Methods sections of scientific publications.…”

Section: Model Implementation: Materials and Methods Recommendationsmentioning

confidence: 99%

Towards community-driven metadata standards for light microscopy: tiered specifications extending the OME model

Hammer

Huisman

Rigano

et al. 2021

Preprint

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While the power of modern microscopy techniques is undeniable, rigorous record-keeping and quality control are required to ensure that imaging data may be properly interpreted (quality), reproduced (reproducibility), and used to extract reliable information and scientific knowledge which can be shared for further analysis (value). In the absence of agreed guidelines, it is inherently difficult for scientists to create comprehensive records of imaging experiments and ensure the quality of resulting image data or for manufacturers to incorporate standardized reporting and performance metrics. To solve this problem, the 4D Nucleome (4DN) Initiative and BioImaging North America (BINA) here propose light Microscopy Metadata specifications that scale with experimental intent and with the complexity of the instrumentation and analytical requirements. They consist of a set of three extensions of the Open Microscopy Environment (OME) Data Model, and because of their tiered nature they clearly specify which provenance and quality control metadata should be recorded for a given experiment. This endeavor is closely aligned with the undertakings of the recently established QUAlity Assessment and REProducibility in Light Microscopy (QUAREP-LiMi; quarep.org) global community initiative. As a result, the ensuing flexible 4DN-BINA-OME (NBO) framework represents a turning point towards increasing data fidelity, improving repeatability and reproducibility, easing future analysis, and facilitating the verifiable comparison of different datasets, experimental setups, and assays. The intention of this proposal is to encourage participation, critiques, and contributions from all imaging community stakeholders, including research and imaging scientists, facility personnel, instrument manufacturers, software developers, standards organizations, scientific publishers, and funders.

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Best practices and tools for reporting reproducible fluorescence microscopy methods

Cited by 81 publications

References 64 publications

Rapid single-molecule characterisation of nucleic-acid enzymes

Rapid single-molecule characterisation of nucleic-acid enzymes

Strain-Level Variation and Diverse Host Bacterial Responses in Episymbiotic Saccharibacteria

Towards community-driven metadata standards for light microscopy: tiered specifications extending the OME model

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