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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.

超分辨率（SR）显微镜是一种前沿方法，能够以高分辨率提供详细的结构信息。然而，样本厚度一直是SR方法的主要限制因素，大型生物结构也带来了挑战。为克服这一问题，关键步骤是优化样本制备，以确保光学均匀性和清晰度，这可以增强SR方法获取较厚结构的能力。卵母细胞是哺乳动物体内最大的细胞，是生殖生物学中的关键研究对象。它们在研究膜蛋白方面特别有用。然而，卵母细胞极其脆弱，对机械操作和渗透压冲击敏感，这使得样本制备成为一个关键且具有挑战性的步骤。我们提出了一种创新、简单且灵敏的卵母细胞样本制备方法，用于三维受激发射损耗（STED）成像采集。这包括酒精脱水并将其置于高折射率介质中。这种扩展的制备程序使我们成功获得了整个小鼠卵母细胞独特的双通道三维STED超分辨率图像。通过优化样本制备，可以克服SR方法当前的局限性，获得大型生物结构（如卵母细胞）的高分辨率图像，以便研究基本生物学过程。

摘要

超分辨率（SR）显微镜是一种前沿工具，使科学家能够观察生物样本中极其精细的细节。然而，对于更大、更厚的样本，它存在困难，因为为了获得最佳成像结果，它们需要在光学上清晰且均匀。在本研究中，我们改进了样本制备过程，使其更适合SR显微镜。我们的方法包括用酒精仔细脱水生物样本，然后将它们转移到增强光学清晰度的封固介质中。这种改进的方案能够对厚生物结构进行高分辨率成像，而这在以前是具有挑战性的。通过优化这种制备方法，我们希望扩大SR显微镜在研究大型生物样本方面的应用，帮助科学家更好地理解复杂的生物结构。