Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Xiaoqing Liu; Bilin Rao; Qingyang Lin; Meiling Gao; Jun Zhang

doi:10.3791/66590

Préparation d’échantillons d’organoïdes rétiniens pour la microscopie électronique à transmission

JoVE Journal
Neuroscience
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JoVE Journal Neuroscience

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Préparation d’échantillons d’organoïdes rétiniens pour la microscopie électronique à transmission

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05:43 min

June 7, 2024

DOI: 10.3791/66590-v

Xiaoqing Liu*^1,2, Bilin Rao*^1,2, Qingyang Lin^1,2, Meiling Gao^1,2, Jun Zhang^1,2

¹State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital,Wenzhou Medical University, ²Laboratory of Retinal Physiology and Disease, School of Ophthalmology and Optometry,Wenzhou Medical University

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Overview

This protocol provides an optimized procedure for preparing retinal organoid samples for transmission electron microscopy (TEM). The study focuses on analyzing the synaptic autostructure in mature retinal organoids, providing a user-friendly method that enables detailed subcellular analysis at the nanoscale level.

Key Study Components

Area of Science

Neuroscience
Retinal Biology
Electron Microscopy

Background

Retinal organoids serve as valuable models for studying retinal structures and functions.
Transmission electron microscopy (TEM) allows for high-resolution imaging of cellular components.
The study aims to improve TEM sample preparation techniques for detailed analysis.
Mature retinal organoids exhibit synaptic structures that can be characterized with this method.

Purpose of Study

To develop a reproducible and straightforward method for TEM sample preparation of retinal organoids.
To evaluate the ultrastructural morphology of synapses within these organoids.
To lay the groundwork for functional investigations and clinical applications.

Methods Used

Utilization of retinal organoids derived from induced pluripotent stem cells (iPSCs).
The protocol involves fixation, staining, dehydration, and embedding processes tailored for TEM.
Key steps include osmium staining, gradient dehydration, and embedding in epoxy resin.
Observations are made using a semi-thin microtome and optical microscopy post-preparation.

Main Results

The method successfully highlights both conventional and ribbon synapses in the retinal organoids.
Ultrastructural morphologies are clearly defined, establishing a solid foundation for further investigations.
The findings support the reproducibility of the method, allowing for subsequent functional studies.

Conclusions

This study enables detailed analysis of synaptic structures within retinal organoids using TEM.
The optimized preparation method facilitates future research in functional retinal organoid studies.
The implications extend to understanding neural mechanisms and potential clinical applications.

Frequently Asked Questions

What are the advantages of using retinal organoids for TEM?

Retinal organoids provide a reproducible model for studying complex retinal structures and are amenable to high-resolution imaging techniques, such as TEM.

How is the sample preparation for TEM implemented?

The sample preparation involves fixation with paraformaldehyde and glutaraldehyde, followed by osmium staining, gradient dehydration, and embedding in epoxy resin.

What types of data can be obtained using this method?

The method allows for detailed imaging of synaptic structures at the nanoscale, enabling investigations of cellular morphology and synaptic organization.

How can this method be adapted for other types of samples?

Similar preparation methods can be adapted for other types of organoids or cell types requiring electron microscopy analysis.

What are the limitations of this TEM preparation protocol?

Potential limitations include the need for specialized materials and equipment, as well as expertise in microscopy techniques for interpretation of results.

Ce protocole fournit une procédure de préparation optimisée et élaborée pour les échantillons d’organoïdes rétiniens pour la microscopie électronique à transmission. Il convient aux applications qui impliquent l’analyse des synapses dans les organoïdes rétiniens matures.

Nos recherches portent principalement sur l’analyse de l’ultrastructure synaptique chez les organoïdes rétiniens matures. Notre objectif est de développer une méthode de préparation d’échantillons de microscope électronique à transmission (MET) reproductible et simple pour les organoïdes rétiniens, ce qui pourrait grandement bénéficier au domaine de la recherche sur les organoïdes rétiniens. Comparé à d’autres techniques telles que le microscope optique, le microscope électronique à transmission offre la possibilité de caractériser l’ultrastructure de la synapse intégrale et les organites subcellulaires dans les organoïdes rétiniens à l’échelle nanométrique.

Notre protocole offre des conditions optimisées pour la préparation d’échantillons de microscope électronique à transmission d’organoïdes rétiniens, en renvoyant des statistiques simples et conviviales. Nos résultats montrent clairement la morphologie ultrastructurelle des synapses au sein des organoïdes rétiniens, y compris les synapses conventionnelles et les synapses en ruban. Cela établit une base solide et présente des méthodes reproductibles pour les investigations fonctionnelles ultérieures des organoïdes rétiniens, ainsi que pour l’évaluation de leurs applications cliniques telles que le criblage de médicaments.

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