Mobile Protein Microboxes on Optical Fibers for Spatial Micromanipulation of Bacterial Microcolonies

Precise spatial control over cell populations offers new opportunities to investigate how physical proximity influences intercellular interactions. While we previously utilized the microfabrication of protein hydrogels for surface patterning, in this work, we present a versatile strategy for actively and reversibly positioning cell populations using protein-based microstructures fabricated by two-photon polymerization. These permeable microboxes are printed directly onto the tips of optical fibers, allowing three-dimensional, micrometer-scale manipulation. Docking with complementary structures prepared on coverslips is enabled via interlocking geometries, notably, a "Christmas tree" design, providing reproducible alignment for long-term mechanical stability. Once docked, the interlocked microbox remains stably engaged on the coverslip, allowing the optical fiber to be withdrawn without perturbing the assembled configuration. Using fluorescently labeled <i>Pseudomonas aeruginosa</i> strains PAO1 and PA14 as a model system, we demonstrate the controlled assembly of distinct bacterial colonies and their precise arrangement within defined microenvironments. This approach provides a generalizable platform for systematic investigation of cellular behaviors, including chemical communication and interspecies signaling with submicrometer spatial precision.