AbstractLive imaging plays a key role in the study of biological processes ranging from the sub-cellular to organism scale, and all the scales between. This requires the development and use of ever evolving innovative microscopies.
The thesis project presented in the following pages describes the further development and use of light-sheet based fluorescence microscopy (LSFM) designed and optimised for imaging the early stages of development of chick embryos. The hardware and software of this microscope were optimised for improvements in performance in data acquisition speed, allowing the complete live 3D sectioning of 4 mm large chick embryos in 2-3 minutes time intervals for periods of up to 20 hours whilst maintaining high spatial resolution. This improved imaging of almost the complete embryo allows the visualisation of the largescale epiblast and hypoblast tissue flows that occur during gastrulation, while providing enough spatial and temporal resolution to be able to detect the detailed behaviours of up to 400 000 cells. Furthermore, the microscope was enhanced to be able to measure at least two fluorophores simultaneously by incorporation of an Optosplit spectral image splitter. This allowed the analysis of two fluorophores in the sample, showing for instance the action and role of two specific proteins or a specific protein and a labelled structure such as the cell membrane, simultaneously.
Further changes on the microscope were implemented in the camera operation included the use of confocal line detection, which greatly reduced background signals and increased image contrast. This improvement allowed the detection of membranous vesicles at the apical side of the epiblast in the space between the embryo and the vitelline membrane. These vesicles were especially prominent in the region where the mesendoderm precursor cells ingress. Confocal line detection allowed closer observation of vesicles formation process, suggesting that the vesicles were released from the apical side of the cells. This led to the hypothesis that when the cells ingress into a future mesendoderm they release these vesicles by an apical abscission process. It remains to be determined whether this is necessary for ingression and what the precise role of these vesicles in the ingression process is. Some of these questions could be resolved with deeper imaging inside the embryo, allowing the ingression process and migration of cells inside the embryo to be followed in more detail. Therefore, efforts were made to improve the imaging resolution deeper in the tissue.
The Gaussian light-sheet focus images the first 1-2 cells layers from the embryo’s surface with acceptable contrast and resolution. Both focus range and penetration depth can, in principle, be increased through beam shaping techniques. Hence, a Bessel beam light-sheet was generated to investigate if it would result in improved penetration depth. The Gaussian beam was modulated into a Bessel beam using a spatial light modulator and the Bessel light-sheet illumination was combined with the confocal line detection mode of the camera. The images acquired were then deconvolved, greatly enhancing image sharpness, although at the cost of a considerably increased image processing time. Embryo surface cellular segmentation analysis showed that the deconvolved Bessel confocal and the Gaussian confocal images were the best illumination modes for Bessel and Gaussian light-sheets, respectively. Overall, embryo surface cellular segmentation was found to be better performed in Gaussian confocal images, showing less segmentation errors when compared to any of the Bessel beam illumination modes or to the Gaussian illumination without confocal line detection mode. However, the image quality deeper into the tissue was not significantly improved with any of the Bessel illumination modes. The main advantage was an extended focus that allows better imaging of curved tissue surfaces that dominate later embryonic development when the embryo starts to fold considerably.
2-photon Gaussian light-sheet illumination readily improved image sharpness at a slightly increased penetration depth. In the case of 2-photon Gaussian light-sheet illumination the main concern is caused by the high photo-damage that results in the physical degradation and death of the embryos in less than 3 hours of live imaging.
Finally, a new microscope was built to investigate whether redundant information provided by combined dual illumination and dual detection light-sheet microscopy can improve image contrast and provide better structural information of the chick embryo. The expectation is that dual illumination will not compromise the sample structure, instead it will optimise the 3D data information and posterior reconstruction, thus minimising the illumination degradation in depth. The microscope has been designed and its construction and validation have started. First proof of principle, chick embryo images have been acquired which highlight the requirement for a meticulous alignment of the microscope optical set-up. Continued work with this microscope is necessary, to reach its optimal operational state.
|Date of Award||2018|
|Supervisor||Kees Weijer (Supervisor) & Michael MacDonald (Supervisor)|
- Chick embryo
- Bessel light-sheet
- 2Photon light-sheet