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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.<\/jats:p><jats:p>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.<\/jats:p><jats:p>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\u2010channel 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\u2010resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes.<\/jats:p><jats:p>Lay Abstract: Super\u2010resolution (SR) microscopy is a cutting\u2010edge 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\u2010resolution 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.<\/jats:p>","DOI":"10.1111\/jmi.13363","type":"journal-article","created":{"date-parts":[[2024,10,11]],"date-time":"2024-10-11T09:27:03Z","timestamp":1728638823000},"page":"165-178","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Innovative sample preparation using alcohol dehydration and high refractive index medium enables acquisition of two\u2010channel super\u2010resolution 3D STED image of an entire oocyte"],"prefix":"10.1111","volume":"297","author":[{"given":"Michaela","family":"Frolikova","sequence":"first","affiliation":[{"name":"Laboratory of Reproductive Biology Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova Vestec Czech 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