Faithful genome transmission in dividing cells relies on correct attachment of all chromosomes to the mitotic spindle. Functional spindles in animal cells require cortical anchorage, maintenance of separate spindle poles, and chromosome attachment to the poles. How microtubules are assembled into such a complex array, wherein distinct microtubule subsets mediate each of these functions, is poorly understood. Here, we aim to elucidate mechanisms underlying spindle assembly using novel microscopy techniques. A scanned Bessel-beam microscope will be built for ultra-fast 3-D live-cell imaging beyond the diffraction limit. This cutting-edge technology will allow us to study, in unprecedented detail, how microtubules originating from centrosomes, kinetochores and the spindle body are organized into the bundles that attach sister chromatid to opposing spindle poles. 3-D structured illumination microscopy will be used to visualize spindle morphology beyond the resolution limit of conventional fluorescence microscopes, allowing us to investigate how post-translational tubulin modifications contribute to the functional diversification of spindle microtubules. Overall, this project will yield insights into the mechanisms underlying spindle assembly and faithful chromosome segregation. Furthermore, implementation of a Bessel beam microscope will contribute to develop the Vienna Biocenter Campus into an internationally leading center for bioimaging.