Exploring Confined 3D Motility of a Microswimmer in Microfluidic Environments

Trypanosoma brucei, the flagellated protozoan parasite responsible for African sleeping sickness, exhibits complex swimming patterns that enable it to navigate heterogenous host environments. To investigate how confinement influences its motility, we developed microfluidic channels with defined geometries of 50 µm × 50 µm and 100 µm × 100 µm (width × height). Using high-speed bright-field microscopy, we recorded trypanosome motion within these controlled environments and generated motion density maps to quantify confinement-dependent spatial exploration. To extend this analysis into three dimensions, we attached submicron tracer particles to individual trypanosomes, allowing depth estimation through defocused diffraction patterns. This integrated microfluidic and optical approach establishes a framework for quantifying T. brucei’s 3D trajectories and understanding how it adapts propulsion strategies to microscale confinement.