Polydiacetylene (PDA) Langmuir films are well known for their blue‐to‐red chromatic transitions in response to a variety of stimuli, including UV light, heat, bio‐molecule bindings, and mechanical stress. In this work, the ability to tune PDA Langmuir films to exhibit discrete chromatic transitions in response to applied mechanical stress is detailed. Normal and shear‐induced transitions are quantified using the Surface Forces Apparatus and established to be binary and tunable as a function of film formation conditions. Both monomer alkyl tail length and metal cations are used to manipulate the chromatic transition force threshold to enable discrete force sensing from ≈50 to ≈500 nN µm−2 for normal loading and ≈2 to ≈40 nN µm−2 for shear‐induced transitions, which are appropriate for biological cells. The utility of PDA thin‐film sensors is demonstrated with the slime mold Physarum polycephalum. The fluorescence readout of the films enabled: the area explored by Physarum to be visualized, the forces involved in locomotion to be quantified, and revealed novel puncta formation potentially associated with Physarum sampling its environment.
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