Higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos
Higher harmonic generation microscopy of in vitro cultured mammal oocytes and embryos
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DOI：
10.1364/oe.16.011574
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发表时间：
2008-07-21
期刊：
影响因子：
3.8
通讯作者：
Sun, Chi-Kuang
中科院分区：
文献类型：
--
作者：
Hsieh, Cho-Shuen;Chen, Shee-Uan;Sun, Chi-Kuang
卵母细胞和胚胎的选择决定着辅助生殖技术的成功。用于选择胚胎的成像工具可能需要包含几个关键特性：非侵入性、高 3D 分辨率和对比度能力，以提供尽可能多的有关胚胎的信息，如纺锤体纤维、透明带和细胞器。目前采用的成像技术只能提供这些所需特性中的一种或两种，并且胚胎的对比度有限。一些成像技术甚至会损坏胚胎。先前的研究表明，谐波发生显微镜（HGM）是一种基于虚拟过渡的技术，可以为斑马鱼胚胎提供亚细胞3D分辨率和毫米级穿透深度的无创成像，因此可能成为未来辅助生殖技术中卵母细胞和胚胎选择的合适工具。然而，为了评估临床应用中的 HGM，必须研究哺乳动物胚胎内二次谐波产生 (SHG) 和三次谐波产生 (THG) 的内在对比起源。在这项工作中，我们结合 SHG 和 THG 模式对体外培养的小鼠卵母细胞和胚胎进行 HGM 研究，重点是对比起源评估。通过无创HGM成像，我们可以清楚地识别整个卵母细胞和胚胎中的各种结构，包括纺锤丝、透明带、极体、细胞膜和细胞内的层状细胞器。通过标准染色研究进一步证实了 THG 对比的起源。通过SHG信号，我们不仅可以观察卵母细胞在中期II停滞时或胚胎卵裂期间的纺锤体纤维，还可以区分和分析透明带三层的厚度。 HGM 结合两种不同的高次谐波产生模式 SHG 和 THG，成功地以高 3D 空间分辨率揭示了整个小鼠胚胎的亚细胞结构。 (c) 2008 年美国光学学会。
Oocyte and embryo selection governs the success of assisted reproductive technologies. The imaging tools applied for selecting embryos may need to contain several key properties: noninvasiveness, high 3D resolution, and the contrast capability to provide as much information about the embryos as possible, such as spindle fibers, zona pellucida, and organelles. Currently adopted imaging techniques can only provide one or two of these desired properties and are with limited contrast of the embryos. Some image techniques can even damage the embryos. Previous studies have shown that harmonic generation microscopy (HGM), a virtual-transition based technology, can provide noninvasive imaging in zebrafish embryos with a sub-cellular 3D resolution and a millimeter penetration depth, and thus could be a suitable tool for future oocyte and embryo selection of assisted reproductive technologies. However to evaluate HGM in clinical use, the intrinsic contrast origin of the second harmonic generation (SHG) and third harmonic generation (THG) inside the mammal embryos has to be studied. In this work we performed HGM studies on the in vitro cultured mouse oocytes and embryos by combining the SHG and THG modalities, with a focus on the contrast origin evaluation. Through the noninvasive HGM imaging, we can clearly identify various structures in the whole oocytes and embryos, including spindle fibers, zona pellucida, polar bodies, cell membranes, and the laminated organelles in the cells. The origin of the THG contrast was further confirmed through the standard staining studies. Through SHG signals, we could not only observe the spindle fibers when the oocytes were arrested at metaphase II or during the cleavage of the embryos, but can also distinguish and analyze the thickness of the three layers of the zona pellucida. Combining two different higher-harmonic generation modalities, SHG and THG, HGM successfully revealed the subcellular structures of the whole mouse embryos with a high 3D spatial resolution. (c) 2008 Optical Society of America.