Dataset DOI: 10.5061/dryad.8kprr4z2z

Description of the data and file structure

Volumetric Electron Microscopy data, in .tiff format, of a centrosome (including both mother and daughter centriole, appendages, subdistal appendages, centrosome-associated dense region, and rootlet) and its corresponding KV cilia.

README — Protocol: Manual 3D segmentation of KV structures in Dragonfly (from vEM stacks)

Purpose: This protocol describes how three-dimensional (3D) segmentations and surface meshes of Kupffer’s vesicle (KV) structures were generated from volumetric electron microscopy (vEM) image stacks using Dragonfly software.

Software: Dragonfly (Object Research Systems)

Input data: vEM image stacks (serial sections) representing the region of interest.

Protocol
1) Import vEM image stacks
- Import vEM-acquired image stacks into Dragonfly.
- Create a volumetric dataset from the imported stack.

2) Manual segmentation (structure tracing)
- For each KV structure of interest:
a) Use Dragonfly segmentation tools to trace the structure across serial sections manually.
b) Continue tracing through the full extent of the structure to produce a complete 3D segmentation.

3) Generate a 3D model and apply a contour mesh
- After generating each structure’s 3D model from the segmentation:
a) Apply a contour mesh to the structure.
b) Use single sampling for x, y, and z coordinates.
c) Set the contour mesh threshold to 50.

4) Smooth the mesh
- Smooth each structure using 2–4 smoothing iterations.
- Adjust the number of iterations until pixelated edge artifacts are eliminated, as confirmed by visual inspection.

5) Assemble the final reconstructed volume
- Generate the final reconstructed volume by aligning individual segmented axes/structures to produce a detailed 3D representation of the sample.

Outputs
- Manual 3D segmentations for each KV structure of interest.
- Contour-meshed and smoothed surface models for each segmented structure.
- A final reconstructed volume combining aligned segmented structures.

Notes
- Smoothing iterations should be chosen empirically (2–4 iterations) based on visual inspection to remove pixelated edge artifacts while preserving the structure geometry.
- The threshold value for contour mesh generation is 50, with single sampling applied uniformly to x, y, and z.

For questions about this protocol or segmentation conventions, refer to the associated manuscript methods or contact the authors.

Files and variables

File: Centrosome_13.tif

Description: One .tif file depicting a centrosome, including appendages, sub-distal appendages, rootlet, CaDr, and mother/daughter centrioles. 

The centrosome begins at slice 2 and (including the cilia) concludes on the final slide. 

File: Concatenated_Stack1_Stack2.tif

This second file is the entire EM data set. The entire KV (slices 1-406) are within this file. It can be accessed via Fiji. All centrosomes were found and processed from here. 

Code/software

Fiji is a free, open-source software package for scientific image processing and analysis.

Access information

Other publicly accessible locations of the data:

• None

Data was derived from the following sources:

• None