Automated Alignment Powered by Computer Vision Streamlines the Two‐Photon Polymerization‐Based Micro 3D Printing of Multiscale and Multimaterial Structures

Two‐photon polymerization (2PP) is a valuable technology for fabricating high‐resolution microstructures. However, the potential for fabricating more complex multimaterial and multiscale microstructures is held back by having to manually align to preexisting structures before printing, which is a time‐consuming and misalignment‐prone process. In this work, we unlock the potential of complex micro 3D printing by proposing a robust and rapid protocol for automated alignment on Nanoscribe Photonic Professional systems. We demonstrate how our protocol streamlines multimaterial, multiscale, and multifile printing, with reduced fabrication time and user workload. The protocol consists of an open‐source program that identifies custom alignment markers via computer vision to adjust the stage positioning. The alignment takes less than 5 s and ensures an overlay accuracy of ≈0.4 μm between different prints. The protocol can be employed for different microscope objectives, substrates, and resins, including smart materials. We expect that our automated alignment protocol will greatly assist Photonic Professional system owners involved with multimaterial and/or multiscale microfabrication. Likewise, we envision that our approach to automated alignment could be adapted to other 2PP systems and potentially other types of resin 3D printers that can execute commands passed as text files.