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A label or watermark does not, by itself, prove that a microscopy image is original. The strongest practical record is an unchanged source acquisition file with its metadata, a separate working copy, and a clear log of acquisition and processing steps. Follow the target journal’s current rules and keep the original available in case editors or reviewers request it.
What makes a microscopy image traceable?
Keep the file produced by the microscope unchanged, then make a copy for processing. The U.S. Office of Research Integrity (ORI) calls the raw image a “safety net”: it allows comparison with later versions and may preserve acquisition conditions stored in a vendor-native format. ORI’s guidance states, “Manipulations of digital images should always be done on a copy of the raw image data.” ORI: Saving the Original
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Maintain a connection between the original, the working copy, and the published figure—for example, through consistent filenames or a separate record. Keep the original format when it contains useful metadata. If conversion is necessary, ORI recommends TIFF and cautions against lossy JPEG for most scientific images. Its older guidance also describes read-only copies and a second copy; the broader principle is redundant storage suited to the size of modern datasets and your retention requirements, not a requirement to use optical discs. ORI: Saving the Original
Do a label or watermark authenticate an image?
No standalone proof is established by the guidance cited here. A visible label can identify a sample, field, or file, and may help prevent mix-ups, but it does not show that the underlying image has not been edited. Embedded metadata and a processing log can support provenance when retained alongside unchanged originals; neither replaces the original acquisition file. This distinction follows from the guidance’s emphasis on preserving source files and documenting processing, rather than from a technical evaluation of watermark systems.
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What acquisition metadata should you retain?
Keep enough information to understand how the image was acquired and, where relevant, to reproduce the experiment. Wiley’s author information recommends reporting the microscope model, objective lenses, detectors, imaging settings such as exposure and laser power, and experimental details such as sample preparation, staining, and environmental conditions. It points authors to the QUAREP-LiMi Bare Minimal Microscopy Reporting Requirements Checklist for standardized reporting. Wiley: Microscopy Research and Technique author information
- Preserve the vendor-native source file and any acquisition metadata it carries.
- Record the microscope and relevant hardware, acquisition settings, and experimental conditions.
- Keep a record of the software and consequential processing steps used to produce the figure.
Which image adjustments should you disclose?
Processing should be minimal, consistent, and transparent. Nature Portfolio’s image standards say to apply whole-image processing equally to controls, avoid contrast changes that make data disappear, and avoid biased thresholding or processing that emphasizes one region at the expense of others. For microscopy, adjustments should cover the entire image; authors should avoid threshold changes and alteration of high signals, and disclose pseudo-color and nonlinear adjustments such as gamma changes. Name the software and explain relevant operations, including deconvolution, 3D reconstruction, rendering, filtering, thresholding, and projection. Nature Portfolio: Image Integrity and Standards (revised 2021-05-01)
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KU Leuven likewise advises disclosing nonlinear adjustments in the legend or representing them with a quantitative color bar. Authors should retain the original unaltered file and be prepared to explain the software and specific tools used. KU Leuven: Guidelines for laboratory and clinical image processing (last updated 2024-02-28)
How should you present crops, composites, and multiple fields?
Make figure construction visible rather than implying that separate material is one continuous acquisition. If a figure combines fields or exposures, mark the boundaries and state the combination in the legend. Explain crops and other consequential assembly choices, and retain the underlying originals so the figure can be checked. Nature Portfolio and KU Leuven both emphasize transparency about image processing and the availability of unaltered files. Nature Portfolio image standards KU Leuven image-processing guidance
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Can a journal ask for raw microscopy files?
Yes, depending on the journal’s policy and the circumstances of evaluation. Nature Portfolio’s standards say editors may request unprocessed data and metadata to assist evaluation, and that evaluation can stall while unavailable source data are resolved. The journal Microscopy, published by Oxford University Press, says manuscripts may be screened by third parties for image alteration and other integrity concerns. That is a policy of that journal, not a universal description of peer review. Check the current instructions for the journal you plan to submit to and be ready to provide source files if requested. Nature Portfolio image standards Oxford University Press: Author guidelines for Microscopy
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A practical recordkeeping workflow
- Save the acquisition original. Keep the unchanged file in its original format, with embedded metadata intact where possible.
- Create a working copy. Perform edits on that copy, not on the only original.
- Record acquisition and processing. Note relevant equipment, settings, experimental conditions, software, and consequential operations.
- Review the figure for fairness. Apply adjustments consistently across the full image and controls; do not selectively conceal or emphasize data.
- Disclose construction and adjustments. Explain nonlinear changes, pseudo-color, composites, and other material processing in the figure or legend as required.
- Check the journal’s current policy. Keep the original files and metadata accessible in case they are requested during evaluation.
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