Ground Truth Protocol Guidelines

Tommy Macrina's Directives (August - September 2025)

I. Mitochondria Segmentation Protocol

1.1 Individual Segmentation Required

Each mitochondrion must be assigned a separate segment ID and color. Do not paint all mitochondria with the same segment.

Reference: Message m10

1.2 Oversegmentation Directive

The original task description stating "do NOT oversegment mitochondria" was incorrect and should be disregarded. Mitochondria must be oversegmented individually.

Reference: Message m8

1.3 Membrane Handling

Do not paint mitochondrial membranes. Leave a visible border/gap around each mitochondrion to represent the membrane boundary.

References: Message m8, Message m15

II. Myelin Segmentation Protocol

2.1 Inner Boundary (Cell Interface)

No padding between myelin and the cell it ensheathes. The myelin segment should directly contact the inner cell.

Reference: Message m15

2.2 Myelin-to-Myelin Contact

No padding between adjacent myelin segments. Myelin segments may touch each other.

Reference: Message m15

2.3 Outer Boundary (Adjacent Cells)

Myelin should approach but not touch the membranes of neighboring cells. Maintain a small gap between myelin exterior and other cell membranes.

Reference: Message m15

III. Upsampled Image Best Practices

3.1 Two-Pass Labeling Approach

Recommended workflow:

1. First pass: Paint annotations on the original image (thicker sections)
2. Second pass: Copy labels onto the upsampled image and connect objects naturally

Reference: Message m2

3.2 Multi-Resolution Viewing

Working exclusively from upsampled images can be misleading. Always cross-reference with original image data.

References: Message m3, Message m4

3.3 Handling Artifacts

"Hallucinations" (artificial structures in upsampled data) should be ignored. Focus on anatomically sensible structures.

References: Message m1, Message m11

IV. Ambiguous Cases & Edge Cases

4.1 Uncertain Organelles

When uncertain whether an object is a mitochondrion (vs. peroxisome, lysosome, etc.), segment it as a mitochondrion. Objects can be removed during review if needed.

Reference: Message m9

4.2 EM Damage Regions

Follow team consensus for handling EM damage. Specific protocols to be determined based on damage type.

Reference: Message m17

V. Workflow & Quality Assurance

5.1 Mandatory Peer Review

Before marking tasks as finished, at least one other team member must inspect your work. Prioritize sharing areas where you struggled.

Reference: Message m13

5.2 Sharing Annotations for Review

Correct procedure in WebKnossos:

1. Navigate to Tasks
2. Search for tasks under the relevant project
3. Click the plus symbol (+) for the task
4. Select "Actions" button (right side)
5. Choose "View"

References: Message m13, Message m14

VI. WebKnossos Tools & Techniques

6.1 Interpolation Tool

The volume interpolation feature is particularly useful for objects perpendicular to the sectioning plane in higher z-resolution data. May require manual cleanup.

Reference: Message m7

6.2 3D Mesh Visualization

• Right-click on any segment to generate a 3D mesh
• Press . (period) to toggle between all views and the view under cursor
• Useful for quality control and identifying errors

Reference: Message m12

6.3 SAM2 AI Segmentation (Experimental)

New AI-assisted segmentation tools available on experimental server:

• Most effective for larger, convex cross-sections without many intercellular objects
• Adjust confidence threshold and erosion parameters for optimal results
• Can use existing segment annotations as hints for consistency

Reference: Message m18

Reference Messages

Below are the complete Slack message threads referenced throughout this document.

Standalone Messages by Tommy Macrina

Threaded Discussions with Tommy Macrina