A sample preparation procedure enables acquisition of 2-channel super-resolution 3D STED image of an entire oocyte

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 larger structures have posed a challenge. To overcome this, the key step is to optimize 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 2-channel 3D STED super-resolution image of an entire mouse oocyte. By optimizing sample preparation, we can overcome the limitations of SR methods and obtain high-resolution images of larger structures, such as oocytes, Knowledge of which are important for understanding fundamental biological processes. RESEARCH HIGHLIGHTS This study aimed to develop a successful sample preparation protocol for imaging mouse oocytes using 3D STED super-resolution microscopy. The results showed that the oocyte sample was optically homogenous, enhancing the capability of the 3D STED method to capture high-resolution images throughout the full depth of the sample, resulting in highly similar SR images. The 3D STED point spread function (PSF) pattern of the depletion laser was successfully secured throughout the entire volume of the sample, including the top and bottom. This study presents the first-ever full volume 3D image of the mouse oocyte, which was acquired using a 2-channel 3D STED method. GRAPHICAL ABSTRACT Introducing an extended sample preparation procedure resulted in an outstanding optical quality sample environment pronounced by high refractive index, high transparency, and minimal spherical aberration. This procedure allowed 3D STED within the entire oocyte.