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JoVE Journal
Neuroscience
Microscopia spettrale riflettometrica su assoni Myelinated In Situ
Microscopia spettrale riflettometrica su assoni Myelinated In Situ
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Microscopia spettrale riflettometrica su assoni Myelinated In Situ

Full Text
7,786 Views
09:13 min
July 2, 2018

DOI: 10.3791/57965-v

Junhwan Kwon1,2, Myunghwan Choi1,2

1Department of Biomedical Engineering,Sungkyunkwan University, 2Center for Neuroscience Imaging Research,Institute for Basic Science (IBS)

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Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study presents a technique for imaging myelinated axons in fixed brain slices utilizing a label-free nanoscale imaging approach based on spectral reflectometry. The method enables analysis of myelin plasticity and demyelination without the need for complex sample preparation.

Key Study Components

Area of Science

  • Neuroscience
  • Axon Imaging
  • Myelin Research

Background

  • Traditional imaging techniques often require complex labeling.
  • Understanding myelin plasticity is crucial for insights into neurodegenerative diseases.
  • The technique can be extended for use in living animals.
  • Label-free approaches minimize disturbances to tissue structure.

Purpose of Study

  • To provide a protocol for studying myelinated axons in fixed tissue.
  • To offer a method for investigating questions surrounding myelin and axonal health.
  • To demonstrate the application of nanoscale imaging in neuroscience research.

Methods Used

  • The primary platform used is spectral reflectometry for imaging.
  • The biological model includes fixed mouse brain slices.
  • No multiomics or metabolic analyses are mentioned in the study.
  • Key steps include tissue fixing and slicing before imaging.
  • Nail polish is used to seal coverslips for preventing contamination.

Main Results

  • SpeRe imaging accurately localized signals along myelinated axons.
  • The method produced results in alignment with traditional fluorescence techniques.
  • No saturation was observed in spectral imaging, validating the reliability of the technique.
  • The imaging allowed for measurement of axon diameter correlating well with fluorescence-based results.

Conclusions

  • This study enables effective imaging of myelinated axons without complex preparations.
  • The technique aids in understanding myelination mechanisms and their plasticity.
  • It presents potential for adaptations in studying living tissues and other neurological questions.

Frequently Asked Questions

What are the advantages of this imaging technique?
The label-free nanoscale imaging technique allows researchers to study myelinated axons without the complications of dye-based labeling.
How is the biological model implemented?
The biological model involves fixing and slicing mouse brain tissue, which is then prepared for imaging using the spectral reflectometry technique.
What types of data are obtained from the imaging?
Data includes the localization of reflectant spectra along myelinated axons and measurements of axon diameter.
How can this method be adapted for living animals?
The protocols can be extended from fixed tissue to living models, enhancing its utility in dynamic studies of myelination.
What are the key limitations of this technique?
Background noise can occur due to the use of silica coverslips, which may affect imaging quality if not properly managed.
What critical steps are involved in the imaging process?
Key steps include glass slide preparation, tissue placement, and careful sealing to prevent contamination before imaging.
Can this technique be used for other types of neural studies?
While specifically aimed at studying myelin, adaptations for other neural structures may be possible, pending validation.

Qui, presentiamo un protocollo passo-passo per l'imaging di assoni myelinated in una fetta di cervello fisso utilizzando una scala nanometrica privo di etichetta tecnica basato su riflettometria spettrale di imaging.

Questa tecnica può aiutare a rispondere a domande chiave nel campo della mielina, come la plasticità della mielina e la demielinizzazione. Il vantaggio principale di questa tecnica è che consente lo studio della nanostruttura dell'assone mielinizzato nel tessuto cerebrale intatto senza alcuna marcatura complessa o preparazione del campione. Qui, dimostreremo l'applicazione in una fetta di cervello, ma questa tecnica può essere estesa agli animali vivi.

Dopo aver fissato e affettato il tessuto cerebrale del topo secondo il protocollo di testo, preparare un vetrino e due vetrini di copertura per ogni fetta di tessuto. Usa un tagliavetro per tagliare a metà uno dei bicchieri quadrati di copertura. Quindi, crea un distanziatore usando la supercolla per attaccare i due pezzi sul vetrino.

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