Feb 16, 2026
Three-Dimensional Neuron Reconstruction from Confocal Z-Stacks (Neurolucida)
- Trinidad Montero1,
- Cristian González-Cabrera2,
- Pablo Henny3
- 1Pontificia Universidad Catolica de Chile;
- 2LIN;
- 3Universidad de Chile
Protocol Citation: Trinidad Montero, Cristian González-Cabrera, Pablo Henny 2026. Three-Dimensional Neuron Reconstruction from Confocal Z-Stacks (Neurolucida). protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp12x8gzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 16, 2026
Last Modified: February 16, 2026
Protocol Integer ID: 243361
Keywords: dimensional neuron reconstruction, labeled neuron, using neurolucida, neurolucida, axonal arbor, dimensional reconstruction, reconstruction
Abstract
This protocol describes three-dimensional reconstruction of single juxtacellularly labeled neurons from confocal z-stack images using Neurolucida. Reconstructions include the somatodendritic domain and axonal arbors, including local collaterals. Serial sections are aligned for continuity, and shrinkage correction along the z-axis is applied as needed to account for histological processing.
Guidelines
4. Trace Axons and Local Collaterals**
- Trace axons from their origin at the soma or proximal dendrites.
- Include local collaterals within the VTA when present.
- Follow long-range axons until they enter major fiber pathways or fluorescence signal becomes insufficient for reliable tracing.
5. Align Serial Sections**
- Use Serial Section Manager to align fragments from each section into a unified 3D reconstruction.
- Verify continuity of processes across section boundaries.
6. Apply Shrinkage Correction**
- Apply correction factors for tissue shrinkage along the z-axis as appropriate for the histological workflow.
- Use a consistent correction method across all reconstructed neurons.
7. Extract Morphometric Parameters**
- Use Neurolucida Explorer to compute morphometric features such as dendritic length, branch points, soma position, and axon length within the traced domain.
Materials
- Confocal microscope capable of z-stack acquisition.
- Image files (z-stacks) covering the neuron across serial sections.
- Neurolucida (MBF Bioscience).
- Serial Section Manager (Neurolucida module).
- Neurolucida Explorer for morphometric analysis.
Troubleshooting
Procedure
Acquire confocal z-stacks across all sections containing the labeled neuron.
Use consistent imaging settings within the dataset.
Ensure sufficient z-step resolution to reliably follow dendrites and axons.
Import z-stacks for each section into Neurolucida.
Define spatial scale parameters using microscope metadata.
Trace soma and dendrites in 3D from z-stacks.
Continue tracing until dendrites reach natural tapering endpoints.
Trace axons from their origin at the soma or proximal dendrites.
Include local collaterals within the VTA when present.
Follow long-range axons until they enter major fiber pathways or fluorescence signal becomes insufficient for reliable tracing.
Use Serial Section Manager to align fragments from each section into a unified 3D reconstruction.
Verify continuity of processes across section boundaries.
Apply correction factors for tissue shrinkage along the z-axis as appropriate for the histological workflow.
Use a consistent correction method across all reconstructed neurons.
Use Neurolucida Explorer to compute morphometric features such as dendritic length, branch points, soma position, and axon length within the traced domain.
Outputs
3D neuron reconstruction files in Neurolucida format.
Traced soma, dendrites, and axons including local collaterals.
Morphometric parameter exports from Neurolucida Explorer.
Critical Steps and Notes
Exclude neurons with incomplete fills.
Maintain consistent tracing criteria across neurons.
Visually confirm accurate alignment of serial sections.
Apply shrinkage correction consistently when comparing morphometrics.