Manual Corrections

Interactive tools for refining automated analysis results.

Overview

While automated segmentation and spot detection are generally accurate, manual correction tools allow you to refine results for maximum accuracy. This is especially important for:

  • Incorrectly split or merged chromosomes

  • False positive spots

  • Ambiguous segmentation boundaries

  • Quality control and validation

The toolkit provides interactive drawing tools integrated with Napari for intuitive corrections.

Manual Correction Workflow

When to Use Manual Corrections:

✅ Segmentation errors (split/merged chromosomes) ✅ False positive spots ✅ Quality control for critical samples ✅ Publication-quality analysis

Recommended Approach:

  1. Run automated analysis

  2. Review results visually

  3. Apply corrections where needed

  4. Save corrections

  5. Re-run intensity calculations

Merging Chromosomes

Use when a single chromosome is incorrectly segmented into multiple regions.

Drawing merge line

Drawing a line to connect regions that should be merged

Step-by-Step Process

1. Prepare the Workspace

Layer visibility

Ensure segmented layer and shapes layer are visible

  • Make sure the segmented layer is visible (eye icon on)

  • Make sure the shapes layer is visible

  • Both layers should be shown simultaneously

2. Select the Shapes Layer

Select shapes layer

Select the shapes layer before drawing

  • Click on the Shapes layer in the layer list

  • It should be highlighted/selected

  • This ensures your drawings go to the correct layer

3. Draw the Merge Line

  • Select the Polygon/Line drawing tool from the top toolbar

  • Click on the first chromosome region you want to merge

  • Continue drawing a line to the second region

  • The line should cross both regions

  • Double-click to finish drawing

Tip

The line doesn’t need to be straight - just make sure it touches both regions you want to merge.

4. Execute the Merge

Click the Merge Chromosomes button

Result:

After merging

Result after merging - regions are now combined

  • The two regions are combined into one

  • They now share the same label/color

  • The shapes layer drawing is removed

  • Segmentation layer is updated

5. Save Your Work

Click Save to preserve the correction

Important

Without saving, the merge will be lost when you load a different image or close the application.

Removing Chromosomes

Use when you want to exclude specific chromosomes from analysis (e.g., edge chromosomes, debris, artifacts).

Drawing removal line

Drawing over a chromosome to mark it for removal

Step-by-Step Process

1. Select the Shapes Layer

  • Click on the Shapes layer in the layer list

  • Ensure it’s highlighted/selected

2. Draw Over the Chromosome

  • Select the Polygon/Line drawing tool

  • Draw a line through the chromosome you want to remove

  • The line must cross or cover part of the chromosome

  • Double-click to finish

Tip

You can draw multiple shapes to mark multiple chromosomes before clicking Remove.

3. Execute the Removal

Click the Remove button

Result:

After removal

Updated segmentation excluding the removed chromosome

  • The marked chromosome(s) are removed from the segmentation

  • The label is set to 0 (background)

  • Other chromosomes remain unchanged

4. Save the Changes

Save button

Save your corrections

Click Save to store the updated segmentation

Note

Saved corrections are loaded automatically next time you open this image set.

Deleting Spots

Use manual spot deletion to remove false positives from either channel.

Deleting Channel 1 Spots (DNA-FISH)

Drawing shapes over spots

Draw shapes over spots to mark them for deletion

Process:

  1. Select the Shapes layer

  2. Draw shapes (rectangles, circles, or polygons) over spots you want to delete

  3. The shape must overlap or cover the spot

  4. Click Delete Channel 1 Spots

Result:

After spot deletion

Updated spot layer after deletion

  • Spots intersecting with drawn shapes are removed

  • The Channel 1 spots layer is updated

  • Shape drawings are cleared

Deleting Channel 2 Spots (CENP-C)

Channel 2 deletion interface

Interface for deleting Channel 2 spots

Same process as Channel 1:

  1. Select Shapes layer

  2. Draw shapes over spots to delete

  3. Click Delete Channel 2 Spots

Result:

Channel 2 after deletion

Channel 2 spots after manual correction

Multiple spots can be deleted at once by drawing multiple shapes or one large shape covering all targets.

Save Spot Corrections:

Click Save to preserve spot deletions

Important

If you don’t save:

  • Corrections are lost when switching images

  • Reloading the image will show original detected spots

  • Batch processing will use original detection

Drawing Tools and Tips

Napari Drawing Tools

Available Tools:

  • Rectangle: Draw rectangular selection boxes

  • Ellipse: Draw circular/elliptical selections

  • Polygon: Draw freeform polygons

  • Line: Draw straight or curved lines

How to Use:

  1. Click the tool icon in the toolbar

  2. Click and drag to draw

  3. Double-click to finish (for polygon/line)

  4. Press Escape to cancel

Tool Shortcuts:

  • Z: Zoom tool

  • P: Pan tool

  • Delete: Remove selected shape

  • Ctrl/Cmd + Z: Undo last action

Drawing Best Practices

For Merging Chromosomes:

  • Draw a clear line connecting both regions

  • Line should touch both chromosomes

  • Doesn’t need to be precise - just connect them

  • Can be straight or curved

For Removing Chromosomes:

  • Line must intersect the chromosome

  • Can draw through multiple chromosomes for batch removal

  • Partial overlap is sufficient

For Deleting Spots:

  • Shape must overlap the spot

  • Drawing a circle/rectangle around spots is easiest

  • Can select multiple spots with one large shape

  • Zoom in for precise selection

Common Drawing Issues

Problem: Shapes appear on the wrong layer

  • Solution: Click the Shapes layer to select it before drawing

Problem: Can’t see the shapes I’m drawing

  • Solution: Toggle the shapes layer visibility (eye icon)

Problem: Double-click doesn’t finish the polygon

  • Solution: Try triple-clicking or pressing Enter

Problem: Accidental shapes drawn

  • Solution: Select the shape and press Delete, or clear all with Clear Shapes button

Saving and Loading Corrections

Saving Corrections

Click the Save button after making any corrections.

What Gets Saved:

  • Updated segmentation masks

  • Modified spot labels

  • Correction timestamps

  • Original files remain unchanged

File Locations:

Corrections are saved as:

` folder_name/ ├── folder_name_segmentation_corrected.npy ├── folder_name_channel1_spots_corrected.npy └── folder_name_channel2_spots_corrected.npy `

Loading Previous Corrections

Automatic Loading:

When you load an image set that has saved corrections:

  • Corrected segmentation loads automatically

  • Corrected spot layers load automatically

  • No need to re-apply corrections

Verification:

Check the console/terminal output:

``` Loading image set: sample_001

  • Found corrected segmentation: sample_001_segmentation_corrected.npy

  • Found corrected Channel 1 spots

  • Found corrected Channel 2 spots

Loaded with corrections.

```

Reverting Corrections

To discard corrections and start over:

  1. Delete the *_corrected.npy files

  2. Reload the image set

  3. Original automated results will load

Correction Workflow for Batch Processing

When batch processing produces results that need correction:

Approach 1: Pre-Correction (Recommended)

  1. Process a few representative images manually

  2. Apply and save corrections

  3. Run batch processing with “Use Saved Results”

  4. Corrected versions will be used automatically

Approach 2: Post-Correction

  1. Run batch processing on all images

  2. Review results and identify images needing correction

  3. Load problematic images individually

  4. Apply and save corrections

  5. Re-run analysis for those specific images

Best Practices

When Making Corrections:

  1. ✅ Zoom in for better precision

  2. ✅ Toggle layer visibility to see clearly

  3. ✅ Save after each correction

  4. ✅ Verify the correction worked before moving on

  5. ✅ Document significant corrections

Quality Control:

  • Review a random sample of automated results

  • Focus corrections on critical samples

  • Keep track of correction frequency (high frequency may indicate parameter issues)

  • Consider adjusting thresholds if corrections are needed often

Time Management:

  • Manual correction takes 1-5 minutes per image

  • Reserve for important samples

  • Use optimized automated parameters for most images

  • Batch process first, then correct outliers

Documentation:

Keep notes on:

  • Which images were corrected

  • Type of corrections made

  • Reasons for corrections

  • Any systematic issues observed

Limitations and Considerations

Subjectivity:

  • Manual corrections introduce subjective judgment

  • Different users may correct differently

  • Establish clear criteria for corrections

  • Consider inter-user validation for publications

Time Investment:

  • Manual correction is time-consuming

  • Not practical for very large datasets

  • Reserve for critical samples

  • Prefer parameter optimization over extensive corrections

Reproducibility:

  • Automated methods are more reproducible

  • Document all manual corrections

  • Save correction files with results

  • Include correction information in methods sections

Next Steps