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Filamentous fungi include serious plant and animal pathogens that explore their environment efficiently in order to
penetrate the host. This environment is physically and chemically heterogeneous and the fungi rely on specific physical
and chemical signals to find the optimal point/s of attack. This study presents a methodology to introduce distinct
structures with dimensions similar to the hyphal diameter and specific chemical surface groups into a controllable
environment in order to study the fungal response. We introduced 3.3 μm polystyrene beads covered with Epoxy surface
groups into microfluidic channels made from PDMS by rapid replica molding. The experimental setup resulted in
different areas with low and high densities of beads as well as densely packed patches. The observations of the fungus
exploring the areas long-term showed that the growth parameters were altered significantly, compared with the values
measured on agar. The fungus responded to both, the physical and chemical parameters of the beads, including
temporary directional changes, increased branching angles, decreased branching distances, decreased apical extension
velocities and occasional cell wall lysis. The wealth and magnitude of the observed responses indicates that the
microfluidic structures provide a powerful platform for the investigation of micron-sized features on filamentous fungi.
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