Preparing Retinal Organoid Samples for Transmission Electron Microscopy

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

doi:10.3791/66590

Vorbereitung von retinalen Organoidproben für die Transmissionselektronenmikroskopie

JoVE Journal
Neuroscience
Author Produced

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Neuroscience

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Vorbereitung von retinalen Organoidproben für die Transmissionselektronenmikroskopie

Full Text

1,597 Views

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

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

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.

Dieses Protokoll bietet ein optimiertes und aufwändiges Präparationsverfahren für retinale Organoidproben für die Transmissionselektronenmikroskopie. Es eignet sich für Anwendungen, die die Analyse von Synapsen in reifen retinalen Organoiden beinhalten.

Unsere Forschung konzentriert sich hauptsächlich auf die Analyse der synaptischen Ultrastruktur in reifen retinalen Organoiden. Unser Ziel ist es, eine reproduzierbare und unkomplizierte Methode zur Probenvorbereitung von Netzhaut-Organoiden (TEM) für Netzhaut-Organoide zu entwickeln, die für das Gebiet der Netzhaut-Organoid-Forschung von großem Nutzen sein könnte. Im Vergleich zu anderen Techniken wie dem optischen Mikroskop bietet das Transmissionselektronenmikroskop die Möglichkeit, die Ultrastruktur der integralen Synapse und der subzellulären Organellen in Netzhautorganoiden auf nanoskaliger Ebene zu charakterisieren.

Unser Protokoll bietet optimierte Bedingungen für die Probenvorbereitung von Netzhautorganoiden durch das Transmissionselektronenmikroskop und liefert einfache und benutzerfreundliche Statistiken. Unsere Ergebnisse zeigen deutlich die Ultrastrukturmorphologie der Synapse innerhalb von retinalen Organoiden, einschließlich der konventionellen Synapse und der Bandsynapse. Damit wird eine solide Grundlage geschaffen und eine reproduzierbare Methode für die anschließende funktionelle Untersuchung von retinalen Organoiden sowie die Evaluation ihrer klinischen Anwendungen wie z.B. das Drug Screening vorgestellt.

View the full transcript and gain access to thousands of scientific videos