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Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue
Published on: July 6, 2011
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3D Reconstruction and Analysis of Thin Subcellular Neuronal Structures using Focused-Ion Beam Scanning Electron
1Division of Sciences & Mathematics, School of Interdisciplinary Arts & Sciences, University of Washington, Tacoma.
Journal of Visualized Experiments : Jove
|October 4, 2021
Summary
This study introduces a new method for 3D analysis of neuronal spinules using focused ion beam scanning electron microscopy (FIB-SEM). The protocol enables rapid visualization and reconstruction of these subcellular structures for research.
Area of Science:
- Neuroscience
- Cell Biology
- Microscopy
Background:
- Advanced scanning electron microscopy enables 3D analysis of subcellular structures.
- Neuronal spinules, thin projections into presynaptic boutons, require detailed nanoscale investigation.
Purpose of the Study:
- To present a methodological pipeline for identifying, visualizing, and analyzing neuronal spinules.
- To enable rapid 3D reconstruction of spinules and associated neuronal components using FIB-SEM.
Main Methods:
- Utilized focused ion beam scanning electron microscopy (FIB-SEM) for high-resolution imaging.
- Developed a decision tree protocol for morphological identification of subcellular structures, including spinules.
- Employed freely available open-source software for data analysis and 3D reconstruction.
Main Results:
- Successfully identified and traced diverse spinules within neuronal synapses.
- Generated 3D reconstructions of spinules, parent neurites, and postsynaptic partners.
- Provided practical tips for improving 3D reconstruction quality for visualization and publication.
Conclusions:
- The presented protocol facilitates rapid nanoscale analysis of subcellular structures within FIB-SEM data.
- This adaptable method serves as an entry point for researchers studying neuronal morphology and synaptic connections.
