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Abstract

Biological systems are first of all physical systems whose function (or malfunction) is also determined by their response to external and internal forces as well as their physical properties. Therefore, measuring mechanical properties is not only essential for a complete understanding of cell function and organism development but also invaluable in disease diagnosis and treatment. Several techniques are available in the field of mechanobiology (an emerging field that focuses on the role of mechanical forces and properties in biology) to measure mechanical properties but they all virtually rely on analyzing the response of the sample to an external perturbation and have limited three-dimensional capabilities. Instead, Brillouin microscopy is a purely optical technique that requires no contact and no external mechanical perturbation of the sample and can provide 3D maps of viscoelastic properties with (sub)micron resolution. Its first demonstration for 3D imaging of biological samples was accomplished in 2007 and, since then, it has gained increasing interest among biologists for its potentialities. In this context, one of the aims of my PhD project was to facilitate the use of Brillouin microscopy for addressing biological questions. I did this by equipping a state-of-the-art Brillouin microscope with the add-ons necessary for studying living biological samples (confocal fluorescence and environment control) and by writing an intuitive software interface that would allow non-experts to use the microscope with minimal training. In collaboration with biologists, we investigated several biological questions, some of which produced interesting findings (that led to scientific publications) while others are still being explored. The second aim of my PhD was to tackle two issues that limit current implementations of Brillouin microscopy: limited speed (leading to imaging times in the excess of hours when whole organisms are imaged in 3D) and potential photodamage, especially in light-sensitive samples. I overcame this limitation by designing a line-scanning version of a Brillouin microscope, with particular attention to ensuring physiological mounting of the sample, low photodamage and high spatial resolution. The speed advantage is given by the parallel acquisition of about 100 Brillouin spectra in a single camera acquisition. This allowed us to image fast processes (gastrulation in Drosophila), large volumes (Phallusia embryos) and follow development of light-sensitive samples (mouse embryos) over two days.