This tutorial guides the process for using the Neuron Annotator plugin for 3D/4D neuron segmentation in Napari.

First-time Usage

Neuron Annotator is a Napari plugin for interactive segmentation using flood filling and watershed algorithms.

Ignore the "Authenticate" window and close it.

  1. Open Napari and load your image
  2. If nnInteractive plugin is opened, close it.
  3. Go to Plugins → Neuron Annotator

Prepare Image Data (Optional)

For 4D Multichannel Data

  1. The plugin displays channel selection checkboxes (Red, Green, Blue)
  2. Select which channels to mix for each color
  3. Click "Synthesize RGB"

Auto Contrast Enhancement

  • Enable "Enhance (Auto Contrast)" to improve visualization
  • Adjust percentile range (default: 1% - 99.9%) if needed

Annotate

Step 1: Add Seeds

  1. Ensure Add (+) mode is selected
  2. Click on the structure you want to segment
  3. The algorithm automatically grows the region based on intensity similarity

Step 2: Adjust Tolerance

  • Use the "Seed Tolerance" slider to control growth aggressiveness
  • Tolerance limits are automatically scaled based on the number of channels
  • For per-seed adjustment: modify the slider and click "Apply to Selected"

Step 3: Remove Overflow

If segmentation grows too much:

  1. Switch to Remove (-) mode
  2. Click on the over-segmented area
  3. Use Local Cut (recommended) — erases a spherical region based on the "Radius" slider

Step 4: Manage Multiple Segments

  • Click "New" to create a new segment
  • Use "Prev" and "Next" to navigate between segments
  • Each segment gets a unique color

Segment Operations:

  • Undo/Redo — Remove or restore the last seed point
  • Reset — Clear all seeds from current segment
  • Delete — Remove entire segment (unless it is the last segment)
  • Merge — Multi-select segments with Ctrl+Click, then click "Merge"
  • Freeze — Lock the current segmentation to prevent further changes
  • Restart — Reload the image and clear all segments

Advanced Features

Edge Constraints

Prevents segmentation from growing across strong intensity gradients.

  1. Enable "Edge Limit" checkbox
  2. Adjust "Edge Threshold" slider (lower = stricter boundaries)
  3. Click "Preview Edges" to visualize detected edges

Note: This feature may not work perfectly in all cases.

Save Your Work

Export Segmentation

  1. Click "Export"
  2. Choose filename (e.g., result.nii.gz)
  3. Creates a NIfTI file with all segments labeled

Fine-Tuning (Optional)

Use Napari's built-in brush/eraser tools on the labels layer for manual corrections.

Note: Save/Load Project has known bugs and is not recommended.

Troubleshooting

  • Segmentation grows too aggressively

    • Lower the Seed Tolerance value
    • Enable Edge Limit for boundary constraints

  • Segmentation does not grow enough

    • Increase the Seed Tolerance value
    • Check that your seed point is placed on a representative pixel

  • Local Cut removes too much or too little

    • Adjust the Radius slider before clicking

  • 4D data not displaying correctly

    • Use "Synthesize RGB" to create a visualization
    • Verify channel selection checkboxes are configured

  • Export fails

    • Ensure you have write permissions to the destination folder
    • Check that at least one segment exists

  • Plugin not appearing in menu

    • Confirm the plugin is installed
    • Restart Napari

  • Other issues

    • Contact the site administrator