STUDY QUESTION Is it feasible to safely determine metabolic imaging signatures of nicotinamide adenine dinucleotide [NAD(P)H] associated auto-fluorescence in early embryos using a light-sheet on-a-chip approach? SUMMARY ANSWER We developed an optofluidic device capable of obtaining high-resolution 3D images of the NAD(P)H autofluorescence of live mouse embryos using a light-sheet on-a-chip device as a proof-of-concept. WHAT IS KNOWN ALREADY Selecting the most suitable embryos for implantation and subsequent healthy live birth is crucial to the success rate of assisted reproduction and offspring health. Besides morphological evaluation using optical microscopy, a promising alternative is the non-invasive imaging of live embryos to establish metabolic activity performance. Indeed, in recent years, metabolic imaging has been investigated using highly advanced microscopy technologies such as fluorescence-lifetime imaging and hyperspectral microscopy. STUDY DESIGN, SIZE, DURATION The potential safety of the system was investigated by assessing the development and viability of live embryos after embryo culture for 67 h post metabolic imaging at the two-cell embryo stage (n = 115), including a control for culture conditions and sham controls (system non-illuminated). Embryo quality of developed blastocysts was assessed by immunocytochemistry to quantify trophectoderm and inner mass cells (n = 75). Furthermore, inhibition of metabolic activity (FK866 inhibitor) during embryo culture was also assessed (n = 18). PARTICIPANTS/MATERIALS, SETTING, METHODS The microstructures were fabricated following a standard UV-photolithography process integrating light-sheet fluorescence microscopy into a microfluidic system, including on-chip micro-lenses to generate a light-sheet at the centre of a microchannel. Super-ovulated F1 (CBA/C57Bl6) mice were used to produce two-cell embryos and embryo culture experiments. Blastocyst formation rates and embryo quality (immunocytochemistry) were compared between the study groups. A convolutional neural network (ResNet 34) model using metabolic images was also trained. MAIN RESULTS AND THE ROLE OF CHANCE The optofluidic device was capable of obtaining high-resolution 3D images of live mouse embryos that can be linked to their metabolic activity. The system's design allowed continuous tracking of the embryo location, including high control displacement through the light-sheet and fast imaging of the embryos (<2 s), while keeping a low dose of light exposure (16 J · cm-2 and 8 J · cm-2). Optimum settings for keeping sample viability showed that a modest light dosage was capable of obtaining 30 times higher signal-noise-ratio images than images obtained with a confocal system (P < 0.00001; t-test). The results showed no significant differences between the control, illuminated and non-illuminated embryos (sham control) for embryo development as well as embryo quality at the blastocyst stage (P > 0.05; Yate's chi-squared test). Additionally, embryos with inhibited metabolic activity showed a decreased blastocyst formation rate of 22.2% compared to controls, as well as a 47% reduction in metabolic activity measured by metabolic imaging (P < 0.0001; t-test). This indicates that the optofluidic device was capable of producing metabolic images of live embryos by measuring NAD(P)H autofluorescence, allowing a novel and affordable approach. The obtained metabolic images of two-cell embryos predicted blastocyst formation with an AUC of 0.974. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION The study was conducted using a mouse model focused on early embryo development assessing illumination at the two-cell stage. Further safety studies are required to assess the safety and use of 405 nm light at the blastocyst stage by investigating any potential negative impact on live birth rates, offspring health, aneuploidy rates, mutational load, changes in gene expression, and/or effects on epigenome stability in newborns. WIDER IMPLICATIONS OF THE FINDINGS This light-sheet on-a-chip approach is novel and after rigorous safety studies and a roadmap for technology development, potential future applications could be developed for ART. The overall cost-efficient fabrication of the device will facilitate scalability and integration into future devices if full-safety application is demonstrated. STUDY FUNDING/COMPETING INTEREST(S) This work was partially supported by an Ideas Grant (no 2004126) from the National Health and Medical Research Council (NHMRC), by the Education Program in Reproduction and Development (EPRD), Department Obstetrics and Gynaecology, Monash University, and by the Department of Mechanical and Aerospace Engineering, Faculty of Engineering, Monash University. The authors E.V-O, R.N., V.J.C., A.N., and F.H. have applied for a patent on the topic of this technology (PCT/AU2023/051132). The remaining authors have nothing to disclose.

中文翻译：

---

使用光片芯片设备对早期胚胎进行代谢成像的新应用：一项概念验证研究。


研究问题：使用光片芯片方法安全地确定早期胚胎中烟酰胺腺嘌呤二核苷酸 [NAD（P）H] 相关自发荧光的代谢成像特征是否可行？摘要答案：我们开发了一种光流体设备，能够使用光片片上设备作为概念验证来获得活小鼠胚胎 NAD（P）H 自发荧光的高分辨率 3D 图像。已经知道的 选择最适合植入和随后健康活产的胚胎对于辅助生殖的成功率和后代健康至关重要。除了使用光学显微镜进行形态学评估外，一种很有前途的替代方案是对活胚胎进行非侵入性成像，以确定代谢活动性能。事实上，近年来，已经使用高度先进的显微镜技术（如荧光寿命成像和高光谱显微镜）对代谢成像进行了研究。研究设计、规模、持续时间通过在双细胞胚胎阶段 （n = 115） 代谢成像后评估胚胎培养后 67 小时的活胚胎的发育和活力来研究该系统的潜在安全性，包括培养条件的对照和假对照（系统未照明）。通过免疫细胞化学评估发育的囊胚的胚胎质量，以量化滋养外胚层和内团细胞 （n = 75）。此外，还评估了胚胎培养过程中代谢活性 （FK866 抑制剂） 的抑制 （n = 18）。 参与者/材料、设置、方法 微结构是按照标准的紫外光刻工艺制造的，将光片荧光显微镜集成到微流体系统中，包括片上微透镜，以在微通道中心产生光片。采用超排卵 F1 （CBA/C57Bl6） 小鼠产生双细胞胚胎和胚胎培养实验。比较研究组之间的囊胚形成率和胚胎质量 （免疫细胞化学）。还训练了使用代谢图像的卷积神经网络 （ResNet 34） 模型。主要结果和机会的作用 光流体设备能够获得活小鼠胚胎的高分辨率 3D 图像，这些图像可以与它们的代谢活动联系起来。该系统的设计允许连续跟踪胚胎位置，包括通过光片的高控制位移和胚胎的快速成像 （<2 s），同时保持低剂量的曝光（16 J · cm-2 和 8 J · cm-2）。保持样品活力的最佳设置表明，适度的光剂量能够获得比使用共聚焦系统获得的图像高 30 倍的信噪比图像（P < 0.00001;t 检验）。结果显示，对照、光照和非光照胚胎（假对照）在囊胚阶段的胚胎发育和胚胎质量方面没有显著差异（P > 0.05;Yate 卡方检验）。此外，与对照组相比，代谢活动受到抑制的胚胎显示囊胚形成率降低 22.2%，代谢成像测量的代谢活动降低 47%（P < 0.0001;t 检验）。 这表明光流体设备能够通过测量 NAD（P）H 自发荧光来产生活胚胎的代谢图像，从而提供了一种新颖且经济实惠的方法。获得的双细胞胚胎代谢图像预测囊胚形成，AUC 为 0.974。大规模数据 N/A. 局限性，谨慎原因 该研究是使用专注于早期胚胎发育的小鼠模型进行的，该模型评估了双细胞阶段的照明。需要进一步的安全性研究，通过调查对活产率、后代健康、非整倍体率、突变负荷、基因表达变化和/或对新生儿表观基因组稳定性的任何潜在负面影响，来评估 405 nm 光在囊胚阶段的安全性和使用。研究结果的更广泛影响这种薄片片上方法新颖，经过严格的安全研究和技术开发路线图，可以为 ART 开发潜在的未来应用。如果展示了完全安全的应用，该设备的整体成本效益制造将有助于可扩展性和集成到未来的设备中。研究资金/利益争夺这项工作得到了美国国家卫生与医学研究委员会 （NHMRC） 的 Ideas Grant（第 2004126 号）的部分支持，得到了莫纳什大学妇产科系生殖与发育教育计划 （EPRD） 以及莫纳什大学工程学院机械与航空航天工程系的部分支持。作者 E.V-O、R.N.、V.J.C.、A.N. 和 F.H. 已就该技术申请了专利 （PCT/AU2023/051132）。其余作者没有什么可披露的。