LAUSR

Optotracers are conformation‐sensitive fluorescent tracer molecules that detect peptide‐ and carbohydrate‐based biopolymers. Their binding to bacterial cell walls allows selective detection and visualisation of Staphylococcus aureus (S. aureus). Here, we investigated the structural properties providing optimal detection of S. aureus. We quantified spectral shifts and fluorescence intensity in mixes of bacteria and optotracers, using automatic peak analysis, cross‐correlation, and area‐under‐curve analysis. We found that the length of the conjugated backbone and the number of charged groups, but not their distribution, are important factors for selective detection of S. aureus. The photophysical properties of optotracers were greatly improved by incorporating a donor‐acceptor‐donor (D‐A‐D)‐type motif in the conjugated backbone. With significantly reduced background and binding‐induced on‐switch of fluorescence, these optotracers enabled real‐time recordings of S. aureus growth. Collectively, this demonstrates that chemical structure and photophysics are key tunable characteristics in the development of optotracers for selective detection of bacterial species.