Wound healing scratch assay analysis.

What is a wound healing scratch assay?

A wound healing scratch assay is an in vitro cell migration experiment. A confluent cell monolayer is scratched to create a cell-free gap, then images are captured over time to estimate how quickly cells migrate into the open area.

The key image-analysis task is to separate the wound region from the cell-covered region, inspect the segmentation boundary, and report measurements that can be traced back to the exact image crop, orientation, and analysis settings used for the experiment.

Which measurements matter?

Wound area percentage

Wound area percentage describes how much of the image field is still open. It is intuitive and commonly reported, but it can be sensitive to crop choices, circular microscope fields, labels, and black borders.

Mean horizontal gap width

Gap width summarizes the wound span across image rows. It is useful when crop or field-of-view differences make area fractions harder to compare. Cytomove reports mean, median, spread, valid rows, and QC warnings so the area and width metrics can be read together.

The Cytomove 1.0 workflow

Cytomove is organized as a step-by-step review workspace instead of a single black-box measurement button. The intended order is:

1. Image QC: load images, create or name groups, crop the field of view, rotate if needed, and exclude unusable images before analysis.
2. Analysis: choose the microscope preset, apply the threshold and variance radius settings, then inspect the calculated wound contours.
3. Manual correction: use fill, erase, undo, and reset only when the automatic mask needs local human review.
4. Publication Figure Builder: assemble a single-group figure, a Control vs Treatment comparison, or a multi-treatment figure from reviewed groups.
5. Export: save metrics, charts, figures, and full-size contour-overlay images for reporting or manual figure assembly.

Image QC before analysis

Image QC is where the raw microscopy view is made consistent before any measurement is trusted. Use it to name the image group, review each time point, set scratch orientation, fine-rotate tilted images, crop the field of view, and remove images that should not be included in the experiment.

The crop is intentionally explicit. If a group has several time points, saving a crop on one image can carry the same crop position forward so the user can keep the field of view consistent across the series rather than redrawing it from scratch each time.

Analysis and contour review

Analysis starts from a preset such as brightfield normal cells, then uses adjustable settings such as threshold, variance radius, minimum region size, and island handling. After clicking Apply, Cytomove displays the calculated contour lines so the user can judge whether the wound boundary is scientifically acceptable before moving on.

Use Apply to group when a setting is appropriate for the whole series. If a publication figure later includes a group that does not yet have stored contours, the Builder shows Analyze missing groups so the selected groups can be completed before figure export.

Manual correction for difficult masks

Manual correction is for edge cases: debris inside the wound, missed wound regions, false-positive cell islands, or local artifacts that the global threshold cannot handle cleanly. It is not meant to replace careful automatic settings; it is a review tool for making a documented correction when the image clearly needs it.

The correction workflow supports fill and erase actions, undo, reset, and visual mask inspection. A good habit is to tune the main analysis settings first, then use manual correction only for the small remaining local issue.

Publication Figure Builder

The Publication Figure Builder turns reviewed groups into presentation-ready summary figures. If only one group is loaded, Cytomove now creates a single-group figure rather than forcing an artificial treatment comparison. With two groups, it builds a Control vs Treatment figure. For drug panels, dose groups, or additional conditions, the multi-treatment template can add extra treatment groups with their own replicate selections.

Panel A shows representative images with the calculated wound contours. Panel B summarizes wound closure, and Panel C can show the normalized wound area or a secondary metric. Titles, labels, fonts, and panel placement can be adjusted in the Builder so the exported figure is closer to a publication layout rather than a raw software screenshot.

How does this relate to ImageJ workflows?

Many wound healing assays are still analyzed with ImageJ macros or manual workflows. Cytomove is designed to complement that familiar approach with a local browser workspace: fast visual review, exportable measurements, and a clear record of the settings used for each image.

Validation against manual and ImageJ-style references is continuing, so Cytomove should be treated as a reviewable academic release tool rather than a substitute for experiment-specific validation.

Supported exports

Cytomove exports both measurement data and figure-oriented assets. The Publication Figure Builder export is designed for manuscript preparation and can include 600 DPI PNG output, TIFF output when supported by the browser environment, PowerPoint/PDF-oriented figure assets, CSV tables, and a Builder ZIP package.

For users who prefer to assemble figures manually, the export package also includes the full-size original images and full-size contour-overlay images. That means the reviewed segmentation result can be reused outside Cytomove without depending only on the compact preview shown in the Builder.

What does browser-local image handling mean?

You can open image files normally through the file picker or drag-and-drop. The current web workflow analyzes those files in the browser and keeps assay image files on your device. This is important for unpublished microscopy data and early-stage academic workflows.