Developmental tissue dynamics using non-diffracting light-sheet microscopy
The development of a new organism is one of the most complex processes in biology. Observing this process in vivo is a prerequisite for many quantitative studies, but requires imaging techniques with a high spatio-temporal resolution that do not affect the development of the sample. Light-sheet based microscopy has become the method of choice, and has allowed new studies of development on a systems level. Nonetheless, imaging of higher organisms remains challenging due to scattering and a limited depth of focus of the illumination light-sheet. In this thesis, a range of methods is discussed to improve the performance of light-sheet microscopes for large and complex developing samples. It was shown that Monte Carlo simulations can not only be used to describe photons, but can also model wave properties like polarisation and interference. These simulations showed that polarisation of the light is an important aspect in label free light-sheet imaging of scattering samples, which was confirmed experimentally. In fluorescence light-sheet microscopy of living chick embryos, non-diffracting Bessel beams gave marginal improvements in penetration depth, and complete threedimensional imaging of the gastrulation phase remained challenging. However, Bessel beams combined with a confocal-line acquisition and a deconvolution improved the images that required a long depth of focus, enabling imaging of the large later stages of chick embryo development. In summary, it was shown that for imaging the highly complex developmental tissue dynamics of the chick embryo, the ideal imaging modality depends on the stage and the curvature of the sample. This requires a flexible lightsheet system with multiple laser lines, with a confocal-line detection, the possibility of non-diffracting illumination and adequate post-processing methods.
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