Cytomove Technical User Guide.

Quick start

Cytomove is organized around a reviewable scratch-assay workflow: Image QC, Analysis, Manual correction, Publication Figure Builder, and Export package. The safest habit is to move through those stages in order and to export only after the contour overlay matches the image you intend to report.

1. Open the Cytomove web app or desktop app.
2. Use Open images, Open image group, drag-and-drop, or + Add another image group to load one or more local images.
3. Name each group clearly, for example HUVEC control r1, HUVEC FDI r2, or Drug B dose 3.
4. Review the first image in Image QC, then set crop, orientation, fine rotation, and exclusion status.
5. Continue to Analysis, choose a microscope preset, click Apply, and inspect the calculated contour overlay.
6. Use Manual correction only when a visible local mask error remains after tuning the main settings.
7. Open Publication Figure Builder, choose single-group figure, Control vs Treatment, or multi-treatment, then update the preview.
8. Export the figure, CSV data, PowerPoint assets, and full-size contour overlays when the preview reflects the reviewed analysis.

Supported inputs

Cytomove currently focuses on local microscopy image files for wound healing scratch assays. Use ordinary image formats such as JPG and PNG. A single file can be reviewed alone, while multiple files can be imported together as a time-series group. When multiple files are selected, Cytomove asks for a group name before creating the group.

Web app and Desktop

The web app is the canonical browser workflow and is available from Open Web App. The desktop app packages the same renderer for local file handling, desktop authentication, update checks, and heavier export use. The core workflow is the same: Image QC, Analysis, Builder, and export.

Access and export permissions

Cytomove allows basic review in the browser, but full CSV, figure data, PNG, and ZIP exports may require a verified sign-in session. The access banner says what is missing: sign-in, email verification, or full access.

Desktop sign-in supports email/password and Google. If an account was created with Google, use Continue with Google rather than the password form. After a successful online approval, desktop can allow a 72-hour offline grace window. If the update policy says the current build is obsolete, a required update gate can block the app until the user downloads or installs the current version.

Image QC

Image QC is the first scientific checkpoint. It defines the image geometry that analysis will use. Use it to choose Single or Group review mode, name groups, switch loaded groups, inspect each time point, set crop, set scratch orientation, rotate images, and exclude unusable images.

Review mode

Review mode has Single and Group modes. Single mode is useful for one image. Group mode is the normal path for a scratch assay series because it displays all time points in the loaded group and makes it easier to check whether 0h, 24h, and 48h are consistent.

Crop, orientation, and rotation

Adjust crop reveals the crop rectangle. Drag the rectangle or its handles until the full wound field is centered, then click Save crop. Reset crop returns to the uncropped image, while Undo returns to the previous crop state when available. A saved crop can carry forward to the next unsaved image so repeated time points stay aligned.

Scratch orientation describes the wound gap direction for analysis. Use vertical scratch for the common left-to-right wound gap and horizontal scratch for top-to-bottom gaps. Fine rotation is for small tilt correction in degrees. Use Rotate left or Rotate right for 90-degree image orientation changes.

Auto crop FOV and exclusion

Auto crop FOV helps remove circular microscope borders or black camera edges from the analysis field. It is useful for brightfield microscope images with a strong field-of-view boundary, but it should still be visually checked. Use Exclude from analysis for blurred images, wrong time points, wrong wells, or fields that should not contribute to group metrics.

Analysis parameters

Analysis converts the reviewed image into a wound mask, contour overlay, and numeric metrics. Start with a microscope preset, click Apply, then evaluate whether the contour follows the biological wound edge. If the settings are good for the whole group, use Apply to group.

Result metrics

Cytomove reports area- and width-based measurements so the wound can be interpreted from more than one angle. Area is intuitive for wound closure, while width is helpful when crop or field-of-view differences make area fractions harder to compare.

• Wound area: open wound pixels after QC crop and segmentation.
• Area fraction: wound area normalized to the usable field of view.
• Wound closure: reduction of wound area relative to baseline.
• Mean gap width: horizontal or vertical gap width summary depending on scratch orientation.
• Normalized area: panel-ready expression of wound area relative to baseline.
• QC warnings: indications such as questionable orientation, missing crop, noisy islands, or incomplete group analysis.

Manual correction

Manual correction is for visible local mask errors after the global analysis settings are already close. It should not replace proper threshold and variance tuning. The correction is strongest when it fixes a small, defensible region that a reviewer could also identify from the image.

• Contour view: preferred for checking whether the calculated wound edge follows the microscopy image.
• Mask view: useful for seeing exactly which pixels are classified as wound.
• Fill area: add a missed wound region when the automatic mask is too conservative.
• Erase scan: remove a false-positive wound region when debris, texture, or cell clusters were misclassified.
• Undo: step back from the most recent correction if the edit was too broad.
• Reset correction: return to the uncorrected analysis result and start correction again.

Publication Figure Builder

Publication Figure Builder creates figure-ready summaries from reviewed groups. It uses stored analysis contours rather than inventing a new contour in the Builder. If a selected group is missing stored contours, the Builder shows Analyze missing groups so those images can be analyzed before export.

Single-group figure

If only one group exists, Cytomove builds a single-group figure. This is useful for a method check, a pilot image series, or a one-condition demonstration. It does not force a Control or Treatment label.

Control vs Treatment

Use Control vs Treatment for a two-condition experiment. Select the Control representative group, Treatment representative group, Control replicates, and Treatment replicates. Panel A shows representative contour overlays, Panel B shows wound closure, and Panel C can show normalized area or another metric.

Multi-treatment

Use multi-treatment when there are more than two conditions: FDI plus another drug, multiple doses, multiple cell treatments, or any condition beyond the first treatment. Each added treatment arm has its own representative group and replicate IDs. Builder plots use dynamic legends so 4+ conditions have reserved x-axis space rather than overlapping labels.

Analyze missing groups

Analyze missing groups appears when Builder selections include groups without complete stored analysis. Click it, wait for analysis to finish, then update the figure. This keeps the Builder preview synchronized with the current Image QC crop and analysis settings.

Builder layout and typography

Builder layout controls are intended for final scientific presentation, not cosmetic screenshot editing. Keep the labels scientifically meaningful and avoid moving panels in a way that hides context.

Validation data and tutorials

Cytomove includes bundled tutorial and validation data so users can test the workflow without uploading their own images first. The full HUVEC tutorial uses the 18-image Control vs FDI validation set, starts in Image QC, teaches crop review, moves to Analysis, adjusts Variance radius, uses Apply and Apply to group, then opens the Builder.

• Full HUVEC validation tutorial: guided 18-image path from Image QC to Builder.
• Manual correction tutorial: teaches reversible fill, erase, undo, and reset correction actions.
• Publication quality figure tutorial: opens the validation set directly in Builder to teach replicate selection, typography, Update Figure, and export.
• Load validation set: available in the Builder controls for testing Control vs FDI with n=3 replicates.

Export package

Cytomove exports both the compact figure and the supporting scientific record. The Builder ZIP is the main publication-oriented package. It can include 600 DPI PNG, TIFF, PowerPoint, CSV tables, figure assets, and full-resolution images.

The full-size contour-overlay files are deliberately separate from the Builder preview. They allow a collaborator, reviewer, or journal workflow to inspect segmentation at the original analysis dimensions.

Parameter glossary

Quality-control checklist

• All images in the group have the correct time point order and group name.
• The crop excludes black borders, labels, and irrelevant microscope field edges.
• Scratch orientation matches the wound geometry before Analysis.
• The first contour is checked visually before Apply to group.
• Manual correction is used only for local visible mask errors.
• Excluded images are intentionally excluded and not accidentally skipped.
• Builder replicate selections match the experimental design and reported n.
• Figure labels, scale bar, p-value label, and Stars annotations are scientifically justified.
• Exported full_images/original and full_images/contour_overlay files are kept with the CSV or Builder ZIP.

Troubleshooting

No images appear after loading

Try a fresh browser tab, then reload the page. If the app was recently updated, a stale browser cache may keep an older script active. The local development server disables stale caching, but public browsers can still hold old files.

Only one image loads from a group selection

Make sure the group picker is being used and that multiple files were selected before confirming the group name. In desktop, the Windows file picker bridges selected files into the same local group loader.

The contour does not match the wound

Return to Analysis, adjust the microscope preset, Threshold level, Variance radius, and Ignore tiny islands mode, then click Apply again. Use Manual correction only after the global settings are close.

The Builder preview says groups need analysis

Use Analyze missing groups. This usually means the group was selected in the Builder before stored contour results existed for all selected images.

Export is disabled

Sign in when full export access is required, verify that selected groups have analysis results, and confirm the Builder preview is available before exporting.

Desktop sign-in does not return from Google

Close the browser tab and try again. The desktop helper waits for the local Google callback on localhost; if another process blocks it, fully close Cytomove and reopen the app.

Desktop asks for an update even after a recent install

Check whether the banner says recommended or required. A recommended update can usually be postponed. A required update means the update policy demands a newer build before analysis can continue.