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Abstract

The dream of creating an accessible neuronal network in a plastic dish has moved closer to realization with the development of stem cell derived brain organoids. In spite of this success, simple technologies for the network characterization in 3D are lacking and thus little is known whether functional networks can be formed. In this thesis, a platform for quantifying electrical activity in 3D neural organoids has been finalized and tested. The setup includes a low-complexity home-built light-sheet microscope that grants single-neuron spatial resolution (<2 μm) at a 3D acquisition speed (5 Hz) compatible with widely used fluorescent Ca2+-sensors. To validate the setup, neural organoids were grown from mouse embryonic stem cells which endogenously express a Ca2+-sensor (RGECO1). The spontaneous network activity was recorded within a field of view of 665 x 665 x 60 μm3. To extract calcium traces of individual neurons, a software package (CaImAn) was used. Spatial and temporal components were decouple using a constrained non-negative matrix factorization of the 4D data. Correlation analysis of the neuronal traces identified clusters with strongly coupled neurons (Spearman’s correlation coefficient > 0.8). The setup will allow studies of how functional neuronal circuits are shaped via self-organization principles of the multicellular system.