When biological systems come in contact with materials, precise control over the materials’ surface properties is required to ensure desired functionality. Polymer brushes are excellent candidates to modify surface properties on demand, finding applications in biosensors, microelectronic parts, tissue engineering substrates, or microfluidic devices [1]. Already established ways to produce such brushes include use of photomasks, direct laser writing and more. However, the achievable pattern resolution is limited. In this work, polymer brushes were photopatterned on confined glass substrates via two photon initiated reversible addition fragmentation chain transfer, 2PRAFT [2]. The biocompatible and hydrophilic monomer N acryloylmorpholine (NAM) was chosen for the synthesis of the polymer brushes. To establish the composition of the polymerisation mixture, the system was initially optimised using RAFT polymerization and blue light irradiation. The glass wafers were covalently modified with RAFT agent 4-cyano-4-(((dodecylthio)carbonothioyl)thio)pentanoic acid (CDTPA) via a two-step procedure, using Ivocerin as the photoinitiator. Firstly, the kinetics of the polymer brushes formation were monitored. Ellipsometry was used for brush thickness measurements, observing a maximum of 10.4 ± 1.5 nm. X ray photoelectron spectroscopy (XPS) was utilised to determine the chemical composition of the brushes. Finally, the established system was further expanded and applied on 2PRAFT, using an established 2P fabrication initiator. The patterned brushes and their morphology were characterised using confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM). The possibilities of this method were further highlighted not only by the ability to print patterns of various colours, but also by the ability to print on vertically stacked surfaces. Funding by the Christian Doppler Research Association within the framework of a Christian Doppler Laboratory for “Advanced Polymers for Biomaterials and 3D Printing” and the financial support by the Austrian Federal Ministry for Digital and Economic Affairs and the National foundation for Research, Technology and Development are gratefully acknowledged. References: [1]Zoppe, J.O.; Ataman, N.C.; Mocny, P.; Wang, J.; Moraes, J.; Klok, H.A. Surface-Initiated Controlled Radical Polymerization: State-of-the-Art, Opportunities, and Challenges in Surface and Interface Engineering with Polymer Brushes. Chem. Rev. 2017, 117, 1105–1318, DOI:10.1021/acs.chemrev.6b00314 [2] Helfert, S.; Zandrini, T.; Rohatschek, A.; Rufin, M.; Machata, P.; Zahoranová, A.; Andriotis, O.G.; Thurner, P.J.; Ovsianikov, A.; Liska, R.; Baudis, S.; Micropatterning of Confined Surfaces with Polymer Brushes by Two‐Photon‐Initiated Reversible Addition–Fragmentation Chain‐Transfer Polymerization. Small Science, 5(1), p.2400263, DOI: 10.1002/smsc.202400263