TY - THES ID - 137382964 TI - Watching blood vessel growth in 3D at the cellular level. Lightsheet imaging for real-time visualization of angiogenesis. AU - Cokelaere, Célie AU - Roeffaers, Maarten. AU - Van Oosterwyck, Hans. AU - KU Leuven. Faculteit Bio-ingenieurswetenschappen. Opleiding Master in de bio-ingenieurswetenschappen. Cel- en gentechnologie (Leuven) PY - 2017 PB - Leuven KU Leuven. Faculteit Bio-ingenieurswetenschappen DB - UniCat UR - https://www.unicat.be/uniCat?func=search&query=sysid:137382964 AB - The process of new blood vessel formation out of already existing vasculature called “angiogenesis” is a crucial process in the development of new tissue and organs, wound healing and a broad range of diseases. To the benefit of humankind, it is essential to fully understand angiogenesis. Research has already proven that the glycolysis is the metabolic pathway responsible for most of the ATP production in endothelial cells. For example, diseases like cancer, resulting in an excess of angiogenesis, can be treated by inhibiting this process. Therefore an inhibitor, 3PO, was determined, which inhibits the rate-limiting step of the glycolysis. During this thesis, a special, truly three-dimensional microscopy technique was established for investigating angiogenesis with high temporal resolution. The advantages of light sheet microscopy are: fast dynamic image acquisition, less photo bleaching and photo toxicity resulting in an excellent tool for time lapse microscopy. After gathering the data, algorithms of traction force microscopy were employed in order to determine the displacements of the beads compared to the reference bead position of the last time point. While imaging, Cytochalasin D (CytoD), an inhibitor of the actin cytoskeleton was used with the purpose to achieve a stress free state of the cells. Angiogenic sprouts have subsequently been examined involving cell culture, light sheet microscopy and algorithms of traction force microscopy. Samples were prepared where human umbilical vein endothelial cells were seeded on a collagen containing fluorescent beads. This gel contains a pro-angiogenic factor as well, which induces the formation of sprouts. Those sprouts were then imaged with a light sheet microscope. There is a strong indication that CytoD can be used to induce the relaxed state in light sheet displacement microscopy. Furthermore, the inhibitor 3PO is likely to indeed have an effect on the cells, due to a decrease in the dynamics of the bead displacements. In the next set of experiments, fast filopodia movement of the sprouts was observed using the advantage of a possible high time resolution with light sheet microscopy. A 20 or 25 μm z-stack was imaged every four seconds. Sprout- and filopodia formation were examined in different collagen concentrations. Especially the length of the sprouts seems to be influenced by the collagen concentration. The filopodia formation and attempts during imaging seemed to have a trend, but only 1 significant result was observed due to the limited number of imaged sprouts per sample. ER -