Thorlabs GmbH 1 article published in JoVE Environment Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales Anna Depetris1, Antoine Wiedmer2, Michael Wagner3, Sebastian Schäfer4, Tom J. Battin1, Hannes Peter1 1Stream Biofilm and Ecosystem Research Laboratory, School of Architecture, Civil and Environmental Engineering, Ecole Polytechnique Fédérale de Lausanne, 2Plateforme technique, School of Architecture, Civil and Environmental Engineering, Ecole Polytechnique Fédérale de Lausanne, 3Water Chemistry and Water Technology, Engler-Bunte-Institut, Karlsruhe Institute of Technology, 4Thorlabs GmbH Microbial biofilms form complex architectures at interphases and develop into highly scale-dependent spatial patterns. Here, we introduce an experimental system (hard- and software) for the automated acquisition of 3D optical coherence tomography (OCT) datasets. This toolset allows the non-invasive and multi-scale characterization of biofilm morphogenesis in space and time.