Document Image Processing Steps for Microscopy Submissions

Updated 4 November 2026 · Standard formula, shown below

Documenting image processing steps is essential for microscopy submissions because journals and reviewers require transparency to validate your results. This guide walks you through creating a clear, reproducible record of every adjustment you make to your images, from acquisition to final figure. You’ll learn what to record, how to organize it, and how to present it in your methods section.

What You’ll Need

The original, unprocessed image files (raw or as exported from the microscope).
The software used for processing (e.g., ImageJ/Fiji, Photoshop, CellProfiler) with version numbers.
A notebook or digital document to log steps as you go.
Access to your institution’s or journal’s specific submission guidelines (they often have a checklist).

Step-by-Step

Record acquisition parameters first. Before any processing, note the microscope type, objective, magnification, numerical aperture, camera model, exposure time, gain, binning, and the file format (e.g., .tif, .czi). This information belongs in your methods section and is critical for reproducibility.
Create a master folder structure. Organize your files into subfolders: one for raw images, one for processed images, and one for final figures. Name files consistently (e.g., sample_condition_rep1_raw.tif).
Open your image in the processing software. Note the software name and version (e.g., Fiji 2.9.0). If you use a script or macro, save it and include it in your documentation.
Document every processing action in order. For each step, write down: what you did (e.g., “applied Gaussian blur with sigma = 2”), the exact settings used, and why you did it (e.g., “to reduce noise before thresholding”). Do this even for steps you think are minor.
Keep a screenshot or log of your processing pipeline. Most software (like Fiji) has a “History” or “Macro recorder” that logs commands. Save this as a text file; it’s an excellent record.
Save processed images as separate files. Never overwrite your raw files. Save the processed version with a clear suffix (e.g., _processed.tif). Use lossless formats like TIFF, not JPEG, unless the journal requires otherwise.
For figures, note any additional adjustments. If you adjust brightness/contrast, crop, or scale the image for the figure, record those steps too. State whether adjustments were linear (e.g., brightness/contrast) or nonlinear (e.g., gamma), as nonlinear adjustments can raise concerns.
Write a methods paragraph. Compile your notes into a concise paragraph describing the processing steps in chronological order, including software versions and settings. Example: “Images were background-subtracted (rolling ball radius 50 px), then a median filter (radius 2) was applied. Brightness/contrast were adjusted linearly for display.”
Include a supplementary file if needed. If your journal allows, upload the full processing log or a screenshot of your pipeline as supplementary material.
Double-check against journal guidelines. Some journals have specific rules (e.g., no nonlinear adjustments, or require stating that “no manipulations were done”). Ensure your documentation aligns.

Common Mistakes

Not recording acquisition settings. Processing documentation is incomplete without knowing how the image was captured.
Overwriting raw files. This is a cardinal sin; you lose the ability to re-process or verify.
Omitting minor steps. Even a simple “crop” or “rotate” should be documented.
Using vague descriptions. “Enhanced contrast” is not enough; specify the method (e.g., “applied contrast stretch from 10th to 90th percentile”).
Forgetting to mention nonlinear adjustments. These can be seen as image manipulation; always disclose them.
Not saving a macro or script. If you repeat the same steps on many images, a script ensures consistency and is easier to document.

Tips

Use a template. Create a spreadsheet or document with columns for step, software, settings, and rationale. This speeds up logging.
Automate with macros. In Fiji, record a macro and run it on all images; then you only need to document the macro itself.
Keep a “processing diary” as you work, not after. It’s easy to forget details.
Be conservative with adjustments. Minimal processing is easier to document and defend.
Check your images at 100% zoom. Ensure no artifacts were introduced by processing.

Troubleshooting

If you forgot to record a step: Re-open the original raw file and try to reproduce the processing. If you can’t, state in your methods that the exact settings are not available, and describe the general approach as best you can. Some journals may ask for a resubmission with proper documentation.
If your journal rejects your image due to “improper manipulation”: Review your documentation to see if you accidentally applied nonlinear adjustments without disclosing. If so, correct the methods and resubmit. If you didn’t, contact the editor to explain your pipeline.
If your processed image looks different from the raw in the final PDF: This may be due to color space conversion. Ensure you save in RGB or grayscale as appropriate and check the PDF.
If you need to re-process after a reviewer request: Use your original raw files and your documented steps. If you have a macro, run it again; if not, follow your notes.

Frequently Asked Questions

Do I need to document every image individually? No, if you process a batch of images identically, you can describe the batch process once. But if any image is processed differently, note that separately.
What counts as image processing vs. manipulation? Processing includes standard adjustments like contrast, brightness, and filtering. Manipulation includes adding or removing features, which is generally not allowed. Always disclose all adjustments.
Can I use Photoshop for microscopy images? Yes, but be cautious. Photoshop is not designed for scientific image analysis, and some journals frown upon it. If you use it, document every step and avoid nonlinear adjustments unless absolutely necessary.
Should I include the software version in my methods? Yes, it’s essential for reproducibility. Versions can change algorithms.
What if I used a third-party plugin? Mention the plugin name and version, and cite the original paper if available.
Is it okay to apply a gamma correction? Yes, but you must disclose it because it’s nonlinear. Explain why it was needed (e.g., to visualize dim signals).

Method: the standard formula for this calculation, rounded as each result states. See how the calculators work or report an error.