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A defensible microscopy submission lets a reader trace every published or measured image back to the microscope’s unaltered output. Keep the original files, work from copies, record the acquisition context, log each consequential operation in order, and disclose any change that could affect interpretation. Put workflow-wide information in Methods and figure-specific details in legends, then apply the destination journal’s current file, source-data, and retention rules.
The goal is not to ban image processing. It is to make processing reproducible and prevent a display choice from being mistaken for a biological finding. The same record should support your own reanalysis months later, a reviewer’s questions, and an editor’s request for source data.
What a complete record must establish
Your documentation should answer five questions without requiring a reader to guess:
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- Which original file produced this figure or measurement? Preserve an unaltered source and maintain a traceable relationship between that source, working files and final outputs.
- What software did you use? Name the acquisition package and every post-acquisition program or plugin that materially changed, measured or rendered the data.
- What operations occurred, in what order and for what reason? Describe steps such as deconvolution, registration, 3D reconstruction, rendering, gamma adjustment, thresholding, segmentation and quantitative measurement rather than saying only that images were “processed.”
- Could the operation change what a reader sees or what a number means? State the adjustment, its scope, whether it was applied consistently, and how the result was calibrated or checked.
The community checklist for quantitative light-microscopy publication treats processing and analysis reporting as part of publishing the data, not as an optional software footnote. Institutional guidance from KU Leuven and the Office of Research Integrity (ORI), along with recommendations from the American Society for Biochemistry and Molecular Biology (ASBMB) and Wiley, converges on this traceability approach. Their scopes differ, so the target journal’s instructions remain controlling.
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Preserve originals and build an image lineage
Keep an immutable source set
Copy the microscope’s original files to controlled storage as soon as practical. Do not overwrite them while adjusting brightness, exporting a new format or testing a segmentation method. KU Leuven and ORI both advise retaining the original and manipulating a copy. If the instrument writes metadata, retain that metadata with the file; exporting to a presentation format can discard acquisition information.
Use a read-only location, access-controlled project folder or other mechanism that makes accidental edits difficult. A backup in a separate storage location protects against disk failure, but a backup is not a substitute for an unmodified source. Keep the original bit depth and file format whenever the instrument or journal permits.
Assign a traceable identifier
Give every acquisition a stable identifier that appears in your internal record and maps to the final figure panel or measurement table. A useful record contains:
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- sample, experiment and acquisition date;
- microscope session or instrument identifier;
- original filename and storage location;
- channel, z-plane or time point where relevant;
- working-copy filename and processing version;
- figure, panel or data-table destination.
Do not rely on a filename such as “final2.tif.” A lineage entry should let another person start with the source and follow each derived file to the submitted output. If one source feeds several panels, record each branch. If a panel combines sources, record every contributing identifier.
Separate analysis data from presentation exports
Keep the data used for measurements distinct from a colorized, downsampled or annotated presentation export. A JPEG or screenshot may be suitable for a submission portal but is generally a poor analysis source because compression and bit-depth conversion can alter intensities. Store the raw or minimally transformed data used for quantification, the parameter file or macro, and the rendered figure as separate artifacts.
Record acquisition context before describing processing
Processing cannot be interpreted without knowing how the signal was acquired. ASBMB’s reporting list includes the following items; Wiley likewise calls for sample, staining, microscope, camera and capture/editing details in Methods or the figure legend.
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| Category | Record | Why it matters |
|---|---|---|
| Sample and preparation | Specimen preparation, fixation or live-imaging conditions, stains and fluorochromes, mounting or imaging medium, temperature and other relevant environmental settings | Signal intensity, background and morphology depend on preparation and environment. |
| Microscope | Make and model; modality where relevant; objective type, magnification and numerical aperture | Optical performance and resolution cannot be inferred from a figure alone. |
| Detector | Camera make and model, detector settings and channel configuration when they affect measurement | Detector response and channel settings influence quantitative values. |
| Acquisition software | Program name and version when available, plus file format and bit depth | The software and encoding determine what metadata and intensity range are retained. |
| Processing software | Each program, plugin or script used after acquisition, with version or release identifier when available | Different implementations can produce different results from the same source. |
| Scale and consistency | Pixel or voxel dimensions, scale bars, and whether compared images were acquired under identical conditions | Readers need a physical reference and a fair basis for comparison. |
Include acquisition settings that materially affect the result, such as exposure, illumination, gain, z-step, time interval or averaging, when your journal or analysis requires them. The cited guidance specifically emphasizes the items in the table; add any instrument parameter needed to reproduce your measurement rather than listing every irrelevant control-panel setting.
Create a processing log that another person can follow
Describe operations in sequence
Write the workflow as an ordered chain from source to output. For each step, state the operation, the input, the important parameters, the reason for using it and the output. For example, “deconvolved” is incomplete; identify the algorithm or software, the measured or theoretical point-spread function if applicable, iteration or regularization settings when they affect interpretation, and whether the result was used for display, measurement or both.
Operations commonly requiring explicit mention include:
- deconvolution or denoising;
- flat-field or background correction;
- registration, alignment or stitching;
- projection, cropping, resampling or downsampling;
- 3D reconstruction, surface rendering or volume rendering;
- channel separation, merging or pseudocoloring;
- linear brightness and contrast adjustments;
- gamma or other nonlinear transformations;
- thresholding, segmentation, object removal or classification;
- intensity, area, length, volume or colocalization measurements;
- annotations, labels and scale-bar creation.
If a step was exploratory but not used in the submitted result, keep it in laboratory records but do not confuse it with the final pipeline. If parameters differed between figures, state the difference and explain why.
Use versioned, reproducible records
Save the processing macro, script, parameter preset or batch-processing log alongside the derived images. Record software versions where the program provides them. A compact log can use columns for source identifier, step number, software and version, operation, parameters, output identifier and analyst. Keep the log with the project rather than in a personal notebook that collaborators cannot access.
When a graphical program does not produce a history, capture the relevant settings in a written record. A screenshot can supplement the record, but it should not replace a readable description in Methods. State whether an operation was applied to the entire image, to every channel, or only to a selected region.
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Distinguish acceptable display adjustments from misleading manipulation
Whole-image adjustments
Adjusting brightness, contrast or color across an entire image can be acceptable when it does not conceal or misrepresent information. Apply the same rule to all images being compared, including relevant controls, and disclose the adjustment. A statement such as “brightness and contrast were adjusted” is stronger when it specifies that the adjustment was applied uniformly to the full frame and whether it was used only for display.
Nonlinear transformations
Gamma and other nonlinear adjustments change the relationship between recorded intensity and displayed intensity. KU Leuven’s guidance says nonlinear adjustments must be disclosed in the figure legend or represented clearly in a quantitative color bar. Name the transformation and identify whether the displayed image was used for measurement. If measurements came from raw data while the figure was gamma-adjusted for viewing, say so explicitly.
Features that must not be selectively changed
KU Leuven states: “No specific feature within an image may be enhanced, obscured, moved, removed, or introduced.” Do not erase a blemish, clone a structure, move an object, add a signal, or selectively sharpen one region to support a conclusion. Cropping can be legitimate for presentation, but retain the full source and show enough context, a scale bar and the crop boundaries or dimensions required by the journal.
Color and channel handling
Explain channel assignments and pseudocolor choices when they affect interpretation. A merged image should make clear which colors represent which channels. Avoid a palette that implies a quantitative scale unless the scale is defined. If a lookup-table change is display-only, identify it; if it changes numerical data, treat it as an analysis operation and document the transformation and calibration.
Keep quantitative analysis tied to raw, calibrated data
ORI advises performing intensity measurements on raw data and calibrating them to a known standard. This means the image used to compute a value should not be a screenshot, contrast-stretched export or selectively enhanced rendering. If preprocessing is necessary for a defined measurement, state exactly what was done before measurement and justify why it preserves the quantity being estimated.
Define selection and exclusion rules
State how fields, cells, particles or time points were selected, including inclusion and exclusion criteria established before analysis where possible. Report whether selection was random, systematic, blinded or convenience-based when that distinction affects bias. Do not present a processed image as representative without explaining how representative fields were chosen.
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Match acquisition conditions for comparisons
ORI recommends acquiring images being compared under identical conditions. If a comparison combines sessions, instruments or settings, disclose the difference and describe normalization or calibration. Include the replicate structure relevant to the analysis: biological versus technical replicates, number of fields or objects, and the statistical approach used to summarize and compare measurements. The image-processing description does not replace a full statistical Methods section; it tells readers how pixels became the reported values.
Preserve calibration and scale
Retain pixel or voxel dimensions and use a scale bar that matches the displayed image. If resampling, stitching or projection changes spatial sampling, record the operation and update the scale metadata or bar. A scale bar copied from an earlier version can become wrong after resizing, so add or verify it after the final geometry is established.
Document composites, projections and rendered views
A figure may combine separate fields, exposures, channels, z-sections or time points. Make the construction visible and explain it in the legend. Mark boundaries between separately acquired fields when they could be mistaken for one continuous image. State whether a panel is a maximum-intensity projection, average projection, orthogonal reconstruction, surface rendering or volume rendering, and identify the source stack and projection method.
Do not imply that a composite represents a single exposure if it does not. If channels were acquired separately, say so. If a montage uses different exposure times or display ranges, identify those differences and explain how comparisons remain valid. Keep each source image and the assembly file in the lineage record.
Place information in Methods and figure legends
Methods: workflow-wide details
Use Methods for information that applies across the study: specimen preparation, stains, microscope and objective, camera, acquisition software, file format and bit depth, processing programs, general pipeline order, calibration, selection criteria, replicates and statistics. Define terms such as “background subtraction” or “segmentation” sufficiently for another laboratory to reproduce the operation.
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Figure legends: panel-specific details
Use each legend for adjustments or constructions unique to that figure: a gamma transformation, a projection type, a different threshold, a channel merge, a crop, a composite boundary or a display-only color map. State when the image is representative and identify the number of independent samples or fields if that information is needed to interpret the panel.
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A concise disclosure pattern
A useful sentence pattern is: “Original 16-bit files acquired with [microscope, objective and camera] were processed in [software and version]. [Operation] was applied to [scope] using [key parameters] for [purpose]. Quantification was performed on [raw or specified data] after [calibration], and the displayed panels received [display adjustment], applied uniformly to [comparison set].” Put variable details inside your manuscript rather than copying a generic claim that does not describe your actual workflow.
Independent reader supportYour contribution helps us test, update, and keep practical guides available for everyone.A practical step-by-step documentation workflow
- Lock the source. Copy original files and metadata to protected storage. Record the acquisition date, sample identifier and original filename.
- Register the acquisition. Enter microscope, objective, numerical aperture, camera, imaging medium, temperature, fluorochromes, acquisition software, file format, bit depth and relevant settings.
- Create a working copy. Give it a new identifier that retains the source identifier. Never edit the source in place.
- Log the first operation. Record the software, version, operation, parameters, purpose and output filename before moving to the next step.
- Continue in order. Add every consequential transformation, including projections, resampling, channel operations, nonlinear adjustments and segmentation.
- Branch deliberately. Keep separate outputs for quantitative analysis and presentation. Record which branch generated each number and panel.
- Check consistency. Verify that compared images were acquired and displayed under comparable rules, and that any difference is disclosed.
- Validate the figure. Confirm scale bars, channel labels, color keys, crop context and composite boundaries after the final resize or layout.
- Write the manuscript description. Put shared workflow details in Methods and exceptions in the relevant legends. Name software and operations instead of using “processed.”
- Audit before submission. Trace every panel and measurement back to an original file, confirm that source data and parameter files are retained, and check the journal’s current upload and disclosure requirements.
Common failure modes and repairs
| Failure | Why it weakens the submission | Repair |
|---|---|---|
| Only “images were processed” appears in Methods | Readers cannot know which operations could affect interpretation. | List software and the ordered operations, with relevant parameters and purpose. |
| Original files were overwritten | There is no verifiable baseline for the figure or measurement. | Recover backups if available; preserve whatever originals remain and document the limitation honestly. |
| Measurements came from a contrast-adjusted export | Display changes can alter intensity values. | Repeat measurements on raw, calibrated data or explain and validate a defined preprocessing step. |
| Gamma adjustment is undisclosed | A nonlinear display can be mistaken for a linear intensity relationship. | Name the adjustment in the legend and provide a quantitative color scale when appropriate. |
| Different fields were merged without boundaries | A reader may interpret a composite as one acquisition. | Mark the boundaries and describe every source and assembly step. |
| Scale bar no longer matches after resizing | Spatial measurements become misleading. | Recalculate or regenerate the bar from final pixel dimensions. |
| Software is named but versions and parameters are absent | Two installations may produce different results. | Add version identifiers and the settings that materially affect the output; retain presets or scripts. |
| One rule is applied to treated images but not controls | Processing can create an apparent group difference. | Apply the same full-image rule to the comparison set or explain a justified exception. |
Journal-specific checks and retention
There is no single universal image-policy rulebook. Before uploading, read the destination journal’s current instructions for accepted file formats, resolution, color mode, source-data files, figure dimensions, naming, raw-data availability and required disclosure location. A journal may request original files separately, require unflattened figures or prescribe how composites and adjustments are described.
Wiley advises retaining original unadjusted data for five years after publication. Treat that as Wiley’s recommendation, not a universal retention period; another journal, funder or institution may require a different period. Keep the source, processing log, scripts or macros, parameter presets and final submitted files for at least the longest applicable requirement, and make sure collaborators know where the archive resides.
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Methods example
“Samples were stained with the indicated fluorochromes and imaged at the stated temperature in the specified imaging medium using a [microscope model] with a [objective magnification and numerical aperture] objective and [camera model]. Images were acquired in [acquisition software and version] as [file format] files at [bit depth]. Original files were retained without alteration. Working copies were processed in [processing software and version] by [ordered operations]. Pixel dimensions and intensity calibration were retained for analysis. Quantification was performed on the raw or specified calibrated data using the predefined selection criteria described below; presentation-only adjustments were applied uniformly to the full comparison set.”
Replace each bracketed item with your actual information; do not leave a generic statement in the manuscript.
Figure-legend example
“Panel A shows a maximum-intensity projection of the recorded z-stack. Channels are displayed with the indicated pseudocolors. Brightness and contrast were adjusted uniformly across the full frame for display; the displayed image was not used for intensity measurement. The merged panel combines separately acquired channels from the same field, and the boundary between stitched fields is marked by a line. A nonlinear gamma adjustment was applied and is stated here; the scale bar represents the final pixel dimensions.”
Adapt the wording to the actual panel. If no gamma adjustment, stitching or projection occurred, omit those clauses rather than implying that they did.
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Final submission checklist
- Original unaltered files and metadata are retained and backed up.
- Every panel and measurement maps to a source identifier.
- Acquisition context includes microscope, objective, numerical aperture, camera, sample, stains, medium, temperature, software, format and bit depth as relevant.
- Processing software, versions and ordered operations are recorded.
- Display-only adjustments are distinguished from analysis transformations.
- Nonlinear changes, projections, renders, crops, composites and channel assignments are disclosed.
- No feature was selectively enhanced, obscured, moved, removed or introduced.
- Measurements use raw or appropriately calibrated data, with selection rules and replicates stated.
- Scale bars and color keys match the final image geometry.
- The destination journal’s current format, resolution, source-data and retention rules have been checked.
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