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.

Centre of the Region Hana for Biotechnological and Agricultural Research Institute of Experimental Botany of the Czech Academy of Sciences Olomouc Czech Republic

Department of Zoology Faculty of Science Charles University Prague Czech Republic

Laboratory of Biomathematics Institute of Physiology of the Czech Academy of Sciences Prague Czech Republic

Laboratory of Integrative Biology Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Laboratory of Reproductive Biology Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova Vestec Czech Republic

Light Microscopy Core Facility Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Schermelleh, L. , Heintzmann, R. , & Leonhardt, H. (2010). A guide to super‐resolution fluorescence microscopy. Journal of Cell Biology, 190(2), 165–175. PubMed PMC

Hell, S. W. , & Wichmann, J. (1994). Breaking the diffraction resolution limit by stimulated emission: Stimulated‐emission‐depletion fluorescence microscopy. Optics Letters, 19(11), 780–782. PubMed

Klar, T. A. , Engel, E. , & Hell, S. W. (2001). Breaking Abbe's diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes. Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics, 64(6 Pt 2), 066613. PubMed

Hell, S. W. (2009). Microscopy and its focal switch. Nature Methods, 6(1), 24–32. PubMed

Klar, T. A. , Jakobs, S. , Dyba, M. , Egner, A. , & Hell, S. W. (2000). Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. PNAS, 97(15), 8206–8210. PubMed PMC

Hein, B. , Willig, K. I. , & Hell, S. W. (2008). Stimulated emission depletion (STED) nanoscopy of a fluorescent protein‐labeled organelle inside a living cell. PNAS, 105(38), 14271–14276. PubMed PMC

Egner, A. , & Hell, S. (2010). Aberrations in confocal and multi‐photon fluorescence microscopy induced by refractive index mismatch. In Pawley J. (ed.), Handbook of biological confocal microscopy (pp. 404–413). Springer.

Diel, E. E. , Lichtman, J. W. , & Richardson, D. S. (2020). Tutorial: Avoiding and correcting sample‐induced spherical aberration artifacts in 3D fluorescence microscopy. Nature Protocols, 15(9), 2773–2784. PubMed

Staudt, T. , Lang, M. C. , Medda, R. , Engelhardt, J. , & Hell, S. W. (2007). 2,2'‐thiodiethanol: A new water soluble mounting medium for high resolution optical microscopy. Microscopy Research and Technique, 70(1), 1–9. PubMed

Egner, A. , Schrader, M. , & Hell, S. W. (1998). Refractive index mismatch induced intensity and phase variations in fluorescence confocal, multiphoton and 4Pi‐microscopy. Optics Communications, 153(4‐6), 211–217.

Flemr, M. , & Svoboda, P. (2011). Ribonucleoprotein localization in mouse oocytes. Methods (San Diego, California), 53(2), 136–141. PubMed

Nagy, Z. , Varghese, A. , & Agarwal, A. (2019). In vitro fertilization A textbook of current and emerging methods and devices: A textbook of current and emerging methods and devices. Springer.

Schatten, H. (2004). Germ cell protocols (vol. 254). Humana Press.

Sarrate, Z. , & Anton, E. (2009). Fluorescence in situ hybridization (FISH) protocol in human sperm. Journal of Visualized Experiments: JoVE, 2009(31), 1405. PubMed PMC

Schindelin, J. , Arganda‐Carreras, I. , Frise, E. , Kaynig, V. , Longair, M. , Pietzsch, T. , Preibisch, S. , Rueden, C. , Saalfeld, S. , Schmid, B. , Tinevez, J.‐Y. , White, D. J. , Hartenstein, V. , Eliceiri, K. , Tomancak, P. , & Cardona, A. (2012). Fiji: An open‐source platform for biological‐image analysis. Nature Methods, 9(7), 676–682. PubMed PMC

Frolikova, M. , Sur, V. P. , Novotny, I. , Blazikova, M. , Vondrakova, J. , Simonik, O. , Ded, L. , Valaskova, E. , Koptasikova, L. , Benda, A. , Postlerova, P. , Horvath, O. , & Komrskova, K. (2023). Juno and CD9 protein network organization in oolemma of mouse oocyte. Frontiers in Cell and Developmental Biology, 11, 1110681. PubMed PMC

Bianchi, E. , Doe, B. , Goulding, D. , & Wright, G. J. (2014). Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature, 508(7497), 483–487. PubMed PMC

Kaji, K. , Oda, S. , Shikano, T. , Ohnuki, T. , Uematsu, Y. , Sakagami, J. , Tada, N. , Miyazaki, S. , & Kudo, A. (2000). The gamete fusion process is defective in eggs of Cd9‐deficient mice. Nature Genetics, 24(3), 279–282. PubMed

Le Naour, F. , Rubinstein, E. , Jasmin, C. , Prenant, M. , & Boucheix, C. (2000). Severely reduced female fertility in CD9‐deficient mice. Science, 287(5451), 319–321. PubMed

Miyado, K. , Yamada, G. , Yamada, S. , Hasuwa, H. , Nakamura, Y. , Ryu, F. , Suzuki, K. , Kosai, K. , Inoue, K. , Ogura, A. , Okabe, M. , & Mekada, E. (2000). Requirement of CD9 on the egg plasma membrane for fertilization. Science, 287(5451), 321–324. PubMed

Aoyagi, Y. , Kawakami, R. , Osanai, H. , Hibi, T. , & Nemoto, T. (2015). A rapid optical clearing protocol using 2,2'‐thiodiethanol for microscopic observation of fixed mouse brain. PLoS ONE, 10(1), e0116280. PubMed PMC

Nieuwenhuizen, R. P. J. , Lidke, K. A. , Bates, M. , Puig, D. L. , Grünwald, D. , Stallinga, S. , & Rieger, B. (2013). Measuring image resolution in optical nanoscopy. Nature Methods, 10(6), 557–562. PubMed PMC

Benammar, A. , Ziyyat, A. , Lefèvre, B. , & Wolf, J.‐P. (2017). Tetraspanins and mouse oocyte microvilli related to fertilizing ability. Reproductive Sciences, 24(7), 1062–1069. PubMed

Valli, J. , Garcia‐Burgos, A. , Rooney, L. M. , Vale De Melo E Oliveira, B. , Duncan, R. R. , & Rickman, C. (2021). Seeing beyond the limit: A guide to choosing the right super‐resolution microscopy technique. Journal of Biological Chemistry, 297(1), 100791. PubMed PMC

Osseforth, C. , Moffitt, J. R. , Schermelleh, L. , & Michaelis, J. (2014). Simultaneous dual‐color 3D STED microscopy. Optics Express, 22(6), 7028–7039. PubMed

Jacquemet, G. , Carisey, A. F. , Hamidi, H. , Henriques, R. , & Leterrier, C. (2020). The cell biologist's guide to super‐resolution microscopy. Journal of Cell Science, 133(11), jcs240713. PubMed

Van Der Wee, E. B. , Fokkema, J. , Kennedy, C. L. , Del Pozo, M. , De Winter, D. A. M. , Speets, P. N. A. , Gerritsen, H. C. , & Van Blaaderen, A. (2021). 3D test sample for the calibration and quality control of stimulated emission depletion (STED) and confocal microscopes. Communications Biology, 4(1), 909. PubMed PMC

Čapek, M. , Janáček, J. , & Kubínová, L. (2006). Methods for compensation of the light attenuation with depth of images captured by a confocal microscope. Microscopy Research and Technique, 69(8), 624–635. PubMed

Barbosa, I. C. R. , Hammes, U. Z. , & Schwechheimer, C. (2018). Activation and polarity control of PIN‐FORMED auxin transporters by phosphorylation. Trends in Plant Science, 23(6), 523–538. PubMed

Kim, J. , Koo, B. K. , & Knoblich, J. A. (2020). Human organoids: Model systems for human biology and medicine. Nature Reviews Molecular Cell Biology, 21(10), 571–584. PubMed PMC