The fluorescein labelled conjugate was subsequently incubated with samples of wild-type E. coli BW25113 and BW25113 ΔbtuB. These samples were then imaged via 3D structured illumination microscopy (3D-SIM) and confocal fluorescence microscopy. Fluorescence signal was observed in WT cells (Figure and) whilst no significant fluorescence was observed in ΔbtuB (Figure ). This shows specific fluorescein labelled conjugate binding to BtuB outer membrane protein on the surface of E. coli cells, whilst providing vital evidence that OPAL ligation does not preclude binding to surface BtuB receptor.