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.
Michaela Frolikova, Michaela Blazikova, Martin Capek, Helena Chmelova, Jan Valecka, Veronika Kolackova, Eliska Valaskova, Martin Gregor, Katerina Komrskova, Ondrej Horvath, Ivan Novotny
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引用次数: 0
Abstract
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.
利用酒精脱水和高折射率介质进行创新性样品制备，可获得整个卵母细胞的双通道超分辨率 3D STED 图像。
超分辨率（SR）显微镜是一种前沿方法，可提供高分辨率的详细结构信息。然而，标本的厚度一直是超分辨显微镜方法的主要限制因素，大型生物结构也是一个挑战。要克服这一问题，关键步骤是优化样品制备，确保光学均匀性和清晰度，从而提高 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，也是生殖生物学的重要研究对象。它们对研究膜蛋白特别有用。然而，卵母细胞非常脆弱，对机械操作和渗透冲击非常敏感，因此样品制备是一个关键且具有挑战性的步骤。我们提出了一种创新、简单而灵敏的方法来制备用于 3D STED 采集的卵母细胞样本。这包括酒精脱水和装入高折射率介质。这种扩展的制备程序使我们成功地获得了整个小鼠卵母细胞的独特双通道三维 STED SR 图像。通过优化样品制备，可以克服目前 SR 方法的局限性，获得大型生物结构（如卵母细胞）的高分辨率图像，从而研究基本的生物过程。论文摘要：超分辨（SR）显微镜是一种尖端工具，可让科学家观察到生物样本中令人难以置信的精细细节。然而，超分辨显微镜在处理较大、较厚的样本时却很吃力，因为样本必须光学清晰、均匀，才能获得最佳成像效果。在这项研究中，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地使生物样本脱水，然后将其转移到能提高光学清晰度的装片介质中。这种改进后的方案能够对厚的生物结构进行高分辨率成像，而这在以前是具有挑战性的。我们希望通过优化这种制备方法，扩大 SR 显微镜在研究大型生物样本方面的应用，帮助科学家更好地了解复杂的生物结构。
期刊介绍：
Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance.
Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.
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