Innovative sample preparation using alcohol dehydration and high refractive index medium enables acquisition of two-channel super-resolution 3D STED image of an entire oocyte.
3D STED
alcohol dehydration
high refractive index medium
large biological objects
oocyte
sample preparation
super‐resolution
Journal
Journal of microscopy
ISSN: 1365-2818
Titre abrégé: J Microsc
Pays: England
ID NLM: 0204522
Informations de publication
Date de publication:
11 Oct 2024
11 Oct 2024
Historique:
revised:
13
09
2024
received:
08
07
2024
accepted:
18
09
2024
medline:
11
10
2024
pubmed:
11
10
2024
entrez:
11
10
2024
Statut:
aheadofprint
Résumé
Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two-channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high-resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes. Lay Abstract: Super-resolution (SR) microscopy is a cutting-edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high-resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures.
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : European Regional Development Fund
ID : CZ.02.1.01/0.0/0.0/16_019/0000827
Organisme : European Regional Development Fund
ID : 68378050-KAV-NPUI
Organisme : European Regional Development Fund
ID : CZ.1.05/1.1.00/02.0109
Organisme : European Regional Development Fund
ID : CZ.02.1.01/0.0/0.0/16_019/0000827
Organisme : MEYS
ID : LM2023050
Organisme : MEYS
ID : CZ.02.1.01/0.0/0.0/18_046/0016045
Organisme : Grant Agency of the Czech Republic
ID : GA-22-30494S
Organisme : Grant Agency of the Czech Republic
ID : GA-21-21736S
Organisme : European Union - Next Generation EU
ID : LX22NPO5102
Informations de copyright
© 2024 The Author(s). Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.
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