无需染色即可实现卵子胚胎的亚细胞级3D成像,突破传统光学限制。
Label-free subcellular 3D imaging of oocytes and embryos via reflection matrix microscopy
- 通过多波长平面波照明采集反射矩阵,计算补偿样本畸变。
- 实现全体积300 nm分辨率的无标记3D成像,穿透致密颗粒细胞层。
- 可清晰识别未成熟卵子的生发泡与核状态,适合临床辅助生殖评估。
非侵入性形态学评估是辅助生殖技术中卵子和胚胎选择的基础,但临床实践仍受限于二维、定性的显微技术。尽管三维荧光成像能提供细胞信息,但其固有的光毒性使其无法用于常规临床。现有无标记方法因两大物理障碍难以解析厚样品中的亚细胞结构:一是包裹卵子的颗粒细胞等大尺度折射率异质性引起的严重像差;二是细胞质脂质等小尺度波动导致的多重散射‘雾化’。本文报道了一种超快速反射矩阵成像(RMI)平台,可克服深度与分辨率限制。通过在多个波长下对一组平面波照明捕获背向散射电磁场,获取多谱段反射矩阵。基于该矩阵,利用数字自适应聚焦算法,计算补偿样本诱导的像差,并重新对齐前向多重散射轨迹与单次散射贡献。该方法实现了卵子与囊胚整个体积内300 nm的前所未有的亚细胞分辨率。我们成功可靠地识别了此前光学手段无法触及阶段中的生发泡与核状态,包括穿透密集颗粒细胞层的成像。该方法为所有着床前阶段提供了强大的非侵入式客观分级工具,可能变革临床体外受精决策流程。
原文摘要 · Abstract (English)
Non-invasive morphological assessment is the cornerstone of oocyte and embryo selection in assisted reproductive technology, yet clinical practice remains limited by two-dimensional, qualitative microscopy. While three-dimensional (3D) fluorescence imaging provides cellular insights, its inherent phototoxicity precludes routine clinical use. Conversely, existing label-free modalities fail to resolve subcellular structures in thick specimens due to two distinct physical barriers: large-scale refractive index heterogeneities, such as the cumulus cells surrounding oocytes, that induce severe aberrations; and short-scale fluctuations, primarily from cytoplasmic lipids, that generate a multiple scattering ``fog''. Here, we report an ultra-fast Reflection Matrix Imaging (RMI) platform designed to overcome these depth and resolution limits. By capturing the back-scattered electromagnetic field for a set of plane-wave illuminations at multiple wavelengths, we record a multi-spectral reflection matrix. From this matrix, we leverage digital adaptive focusing algorithms to computationally compensate for sample-induced aberrations while realigning forward multiple scattering trajectories with the single-scattering contribution. This approach enables label-free 3D visualization of oocytes and blastocysts with an unprecedented subcellular resolution of 300 nm throughout the entire specimen volume. We demonstrate the reliable identification of germinal vesicles and nuclear status in stages previously inaccessible to conventional optics, including imaging through dense cumulus cells. Our method provides a powerful, non-invasive tool for objective grading across all pre-implantation stages, potentially transforming decision-making in clinical IVF.
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