Faculty of Biology-Oriented Science and Technology, Kindai University, Kinokawa, Wakayama, Japan.
Faculty of Life and Environmental Science, University of Yamanashi, Kofu, Yamanashi, Japan.
Methods Mol Biol. 2025;2872:21-35. doi: 10.1007/978-1-0716-4224-5_2.
Live cell imaging techniques are now essential for capturing chromosomal segregation in fertilized eggs. Although better spatiotemporal resolution of fluorescence observations could provide more information, higher phototoxicity may occur. Super-resolution microscopy is generally considered unsuitable for live cell imaging because of the considerable cell damage. Here, we developed a method for counting chromosomes in mouse living oocytes and early embryos using super-resolution microscopy based on disk confocal photon reassignment microscopy (OPRA). In this chapter, we describe the imaging conditions for minimally invasive, high-resolution observation of mouse oocytes and embryos and a method to count chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization.
活细胞成像技术现已成为捕获受精卵中染色体分离的重要手段。尽管荧光观察的更高时空分辨率可以提供更多信息，但可能会出现更高的光毒性。超分辨率显微镜通常被认为不适合活细胞成像，因为它会对细胞造成相当大的损伤。在这里，我们开发了一种使用基于盘状共聚焦光子重分配显微镜（OPRA）的超分辨率显微镜对小鼠活体卵母细胞和早期胚胎进行染色体计数的方法。在本章中，我们描述了用于对小鼠卵母细胞和胚胎进行微创、高分辨率观察的成像条件，以及一种通过 CRISPR/dCas 介导的活细胞荧光原位杂交进行染色体计数的方法。
