abberior dyes & labels
2024
Journal of Microscopy
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
Authors:
Michaela Frolikova, Michaela Blazikova, Martin Capek, Helena Chmelova, Jan Valecka, Veronika Kolackova, Eliska Valaskova, Martin Gregor, Katerina Komrskova, Ondrej Horvath, Ivan Novotny
Keywords:
alcohol dehydration; 3D STED; high refractive index medium; large biological objects; oocyte,sample preparation; super-resolution
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.