Development of lightsheet microscopy to study gastrulation in amniote embryo'

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.