Fig. 6: Species-specific probes can be used for simultaneous species and ribosome phenotype phenotype identification.

a A one-to-one mixed sample of fixed and permeabilised E. coli and P. aeruginosa cells was mixed and suspended in hybridisation buffer with species-specific FISH probes targeting the 16S rRNA. The E. coli cells had been treated with 1 × EUCAST ciprofloxacin for 30 min to develop a ciprofloxacin-treated ribosome phenotype. The E. coli-specific probe was labelled with an ATTO-532 fluorophore while the P. aeruginosa-specific probe was labelled with a Cy5 fluorophore. After the sample was imaged on a fluorescence microscope, the E. coli cells were segmented and passed to the ciprofloxacin phenotype classifier, which identified them as ciprofloxacin-treated with 97.1% accuracy. b The E. coli cells can be visualised with the ATTO-532 fluorophore (green), illuminated with 532 nm excitation. c The P. aeruginosa cells can be visualised with the Cy5 fluorophore (magenta), illuminated with 647 nm excitation. There is some signal in the E. coli cells, at approximately half the brightness of the P. aeruginosa cells. d The composite image showing both ATTO-532 (green) and Cy5 (magenta) channels. The scale bar (white) is 2 μm.