High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

Luca Zaeck; Madlin Potratz; Conrad  M. Freuling; Thomas M&#252;ller; Stefan Finke

doi:10.3791/59402

Imagerie 3D haute résolution de l'infection par le virus de la rage dans un tissu cérébral éclair...

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Neuroscience

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JoVE Journal Neuroscience

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

Imagerie 3D haute résolution de l'infection par le virus de la rage dans un tissu cérébral éclairci par solvant

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09:42 min

April 30, 2019

DOI: 10.3791/59402-v

Luca Zaeck¹, Madlin Potratz¹, Conrad M. Freuling¹, Thomas Müller¹, Stefan Finke¹

¹Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut,Federal Research Institute for Animal Health

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Overview

This study explores novel immunostaining-compatible tissue clearing techniques that enable 3D visualization of rabies virus brain infection and its intricate cellular environment. The method enhances imaging depth, facilitating thorough analysis with confocal laser scanning microscopy.

Key Study Components

Area of Science

Neuroscience
Immunology
Tissue Imaging

Background

Understanding viral brain infections is crucial for neuroscience research.
Existing imaging techniques often face challenges with tissue opacity.
Immunostaining can differentiate various cellular subpopulations.
Recent advances in tissue clearing have improved imaging capabilities.

Purpose of Study

To visualize rabies virus infection in brain tissues.
To analyze the spatiotemporal resolution of the infection environment.
To enhance capabilities of deep tissue imaging through immunostaining.

Methods Used

The study utilizes immunostaining-compatible tissue clearing techniques.
Mouse brain tissue samples are employed as the biological model.
Immunostaining procedures allow visualization of virus phosphoprotein layers.
Various incubation and washing steps are conducted for optimal imaging.
The cleared tissues are captured using confocal laser scanning microscopy, enabling 3D analysis.

Main Results

The method successfully visualizes layers of infected neuronal cells.
High-resolution imaging correlates antigen abundance and distribution within cells.
Seamless 3D projections showcase the complexity of viral infections.
Detailed insights enhance understanding of infection processes at the cellular level.

Conclusions

This study demonstrates an effective technique for visualizing viral brain infections.
It underlines the importance of immunostaining in facilitating deep tissue imaging.
Findings contribute to a better understanding of neuronal mechanisms in response to infections.

Frequently Asked Questions

What are the advantages of the immunostaining-compatible clearing method?

This method allows for deep tissue imaging while preserving cellular structures, enabling detailed analysis of complex environments sparked by infections.

How is the rabies virus infection simulated in this study?

Infection is simulated through the introduction of the virus into mouse brain tissue, followed by extensive immunostaining procedures to reveal cellular interactions.

What types of data can be obtained through this method?

The method provides high-resolution images that reveal the abundance and distribution of viral antigens and cellular subpopulations within the brain tissue.

How can this method be applied in future research?

This approach can be adapted for studying other viral infections or diseases affecting the brain, allowing researchers to visualize interactions at a cellular level.

What are the limitations of the tissue clearing technique?

The technique may require optimization for different types of tissues or pathogens, and the extensive duration of the imaging process can limit throughput.

Les nouvelles techniques de compensation des tissus immunostaining-compatibles comme l'imagerie 3D ultime des organes éclaircis par solvant permettent la visualisation 3D de l'infection cérébrale du virus de la rage et de son environnement cellulaire complexe. D'épaisses tranches de tissu cérébral étiquetées par anticorps sont rendues optiquement transparentes afin d'augmenter la profondeur d'imagerie et de permettre l'analyse 3D par microscopie de balayage laser confocal.

Ce protocole permet la visualisation de l’infection virale et fournit des aperçus profonds de la résolution spatiotemporale de l’environnement de l’infection et de son contexte cellulaire environnant. L’applicabilité de l’immunostaining à l’imagerie tissulaire profonde permet non seulement la détection de virus, mais permet en fait également la différenciation de diverses sous-populations cellulaires à l’aide de leurs marqueurs cellulaires respectifs. Pour fixer le tissu pour l’immunostaining placer les échantillons de cerveau dans un rapport un à 10 de 4%paraformaldéhyde dans PBS pour échantillonner le volume de tissu pendant au moins 48 heures à quatre degrés Celsius.

À la fin de la période de fixation, laver les échantillons de tissus trois fois dans le PBS pendant au moins 30 minutes par lavage avant de transférer les échantillons à 02 % d’azide de sodium dans le PBS à quatre degrés Celsius. Ensuite, utilisez un vibratome réglé à un taux d’alimentation de 0,3 à 0,5 millimètre par seconde pour sectioner les tissus en sections d’un millimètre d’épaisseur. Conservez les sections dans de l’azide de sodium frais à 02 % en PBS à quatre degrés Celsius.

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