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Dedicated Setup for the Photoconversion of Fluorescent Dyes for Functional Electron Microscopy
Katharine L Dobson1,2, Carmel L Howe3, Yuri Nishimura1,4
1Department of Neuroscience, Psychology and Behaviour, University of Leicester, Leicester, United Kingdom.
Frontiers in Cellular Neuroscience
|August 17, 2019
Summary
We developed a cost-effective setup for photoconversion, turning fluorescent signals into electron-dense ones for detailed neuronal imaging. This method enhances reliability and throughput for studying neuronal morphology and function.
Area of Science:
- Neuroscience
- Microscopy
- Cell Biology
Background:
- Studying fine neuronal structures requires advanced imaging techniques.
- Correlative Light and Electron Microscopy (CLEM) offers high-resolution insights.
- Photoconversion of fluorescent signals to electron-dense ones is crucial for CLEM.
Purpose of the Study:
- To describe a cost-effective and improved setup for targeted photoconversion.
- To enhance the reliability and throughput of DAB oxidation for neuronal imaging.
- To facilitate the study of neuronal morphology and function in thick tissue samples.
Main Methods:
- Development of a simple, cost-effective photoconversion setup.
- Utilizing localized oxidation of diaminobenzidine (DAB) mediated by excited fluorophores.
- Application of Correlative Light and Electron Microscopy (CLEM) for visualization.
Main Results:
- The setup simplifies the photoconversion workflow, increasing photoillumination depth.
- Improved targeting and reduced processing time for individual samples.
- Successful demonstration with FM 1-43FX and Lucifer Yellow in cortical slices.
Conclusions:
- The described setup offers a practical solution for enhanced photoconversion in neuroscience research.
- It is particularly effective for thick neuronal tissues, overcoming light penetration and oxygen diffusion limitations.
- The system is adaptable for various dyes and nanoparticles capable of DAB oxidation.
Keywords:
CLEMFM1-43Lucifer Yellowcorrelative light electron microscopyelectron microscopyfunctional labelingsynapsesynaptic vesicle (SV)More Related Videos
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