Development of lightsheet microscopy to study gastrulation in amniote embryo'

原肠发育是脊椎动物胚胎早期发育的重要阶段。在原肠形成过程中，胚胎具有其特有的三层组织结构。原肠发育包括协调细胞分裂、细胞分化和胚胎中数十万个细胞的高度有组织的大规模运动。我们研究了鸡胚胎中这些关键细胞行为是如何通过化学和机械细胞-细胞信号整合在一起的，鸡胚胎是人类发育的优秀模型系统[1]。这项分析需要将胚胎在正常和实验扰动条件下发育过程中的这些关键细胞行为可视化。这已经成为可能，因为开发了强大的新的遗传和转基因方法来荧光标记转基因鸡系中特定的细胞结构和细胞类型，以及专门的实时成像方法，如光片显微镜，以更长时间对细胞和组织动态进行成像。光片显微镜能够有效地对荧光标记的生物组织进行光学切片，以最小的光损伤产生整个胚胎的高对比度、高分辨率图像，这对胚胎发育的长期成像至关重要[2]。我们正在使用新的转基因小鸡品系，并设计和建造了一种专用的光片显微镜，使我们能够在胚胎中以高空间和时间分辨率成像这些复杂的细胞行为，为它们的发育提供新的见解[3]。为了充分利用光片显微镜的潜力，有必要根据正在研究的生物样本(这里是鸡胚)定制这些仪器。这需要光学、机械仪器、专门的样本室的紧密集成，与样本培养和生存要求带来的生物限制相兼容，以及大规模计算图像数据采集和分析[3]。该项目的目标是：1)进一步调整和优化现有的光片显微镜，包括改进的自动样品跟踪、照明(光束成形)模式、样品处理以及数据采集和分析的速度。2)利用这些改进的显微镜对正常和扰动实验条件下的细胞分裂、分化和细胞运动进行量化，以进一步阐明在时空胚胎发育过程中控制这些复杂细胞行为协调的基本原理。拟议项目的主要新颖方面是：目前的光片显微镜系统是顶视显微镜，我们现在的目标是建造一种倒置光片显微镜，使其他实验技术(如激光切割和光学镊子)能够更容易地获取样品，并方便样品处理[4]。我们建议采用新的低折射率聚合物来开发用于鸡胚胎成像的折射率匹配样品室[5]。LightSheet显微镜产生非常大的图像数据集(&gt；4TB/实验)。大规模新型图像处理和数据分析方法的应用和进一步发展将是该项目的重要组成部分[6]。这个项目需要有物理/工程和计算技能(例如，MatLab/Phyton，C++)的背景，对生命科学感兴趣，并有能力在高度跨学科的环境中工作。它提供了参与生物实验和相关数据分析的独特机会。参考文献1.Serrano Najera，G.和C.J.Wejer，驱动鸟类胚胎原肠形成的细胞过程。《机械开发》，2020。163.103624页。2.万，Y.，K.麦克多尔和P.J.凯勒，光片显微镜及其理解发育过程的潜力。Annu Rev Cell Dev Biol，2019年。35：第655-681页。

Gastrulation is an essential phase in the early embryonic development of all vertebrate embryos. During gastrulation the embryo takes on its characteristic three layered organisation. Gastrulation involves the coordination of cell divisions, cell differentiation and highly organised large scale movements of hundreds of thousands of cells in the embryo. We study how these key cell behaviours are integrated by chemical and mechanical cell- cell signalling in the chick embryo an excellent model system for human development [1]. This analysis requires the visualisation of these critical cell behaviours during the development of the embryo under normal and experimentally perturbed conditions. This has become possible through the development of powerful new genetic and transgenesis methods to fluorescently label specific cellular structures and cell types in transgenic chick lines as well as dedicated live imaging methods such as lightsheet microscopy to image cell and tissue dynamics for extended periods of time. Lightsheet microscopy has the ability to efficiently optical section fluorescently labelled biological tissues to produce high contrast, high resolution images of whole embryos with minimal photo-damage, critical for long term imaging of embryonic development [2]. We are using new transgenic chick lines and have designed and built a dedicated lightsheet microscopes that allows us to image these complex cell behaviours at high spatial and temporal resolution in embryos providing new insights in their development [3]. To take full advantage of the potential of lightsheet microscopy it is necessary to customise these instruments to the biological sample (here, chick embryos) being studied. This requires a close integration of optics, mechanical instrumentation, specialised sample chambers, compatible with biological constraints posed by sample culture and survival requirements as well as largescale computational image data acquisition and analysis [3]. The aims of this project are: 1) To further adapt and optimise existing lightsheet microscopes, including improved automatic sample tracking, illumination (beam shaping) modes, sample handing and speed of data acquisition and analysis. 2) To use these improved microscopes to quantify cell division, differentiation and cell movements under normal and perturbed experimental conditions to further elucidate the fundamental principles that control the coordination of these complex cell behaviours during embryogenesis in space-time. Key novel aspects of the proposed project are: Current lightsheet microscopy systems are top view microscopes, we now aim to build an inverted lightsheet microscope, to allow easier sample access for additional experimental techniques, such as laser cutting and optical tweezing and facilitate sample handling [4]. We propose to adapt new low refractive index polymers for the development of refractive index matched sample chambers for the imaging of chick embryos[5].. Lightsheet microscopes generate very large image data sets (>4TB/experiment). Application and further development of large scale novel image processing and data analysis methods will be an essential part of this project [6]. This project requires a background in physics/engineering and computational skills (e.g. Matlab/Phyton, C++) and an interest in life sciences and ability to work in a highly interdisciplinary environment. It offers unique opportunities to participate in biological experiments and associated data analysis. References 1. Serrano Najera, G. and C.J. Weijer, Cellular processes driving gastrulation in the avian embryo. Mech Dev, 2020. 163: p. 103624. 2. Wan, Y., K. McDole, and P.J. Keller, Light-Sheet Microscopy and Its Potential for Understanding Developmental Processes. Annu Rev Cell Dev Biol, 2019. 35: p. 655-681.