Figure 6: Dual colour dSTORM of Salmonella using fusion proteins to HaloTag and SNAP-tag.

Flagellar motor subunit FliN and SPI1-T3SS subunit SpaS were visualized within one cell. (A) FliN-SNAP-tag and SpaS-HaloTag were stained with 30 nM TMR-Star (green) and 150 nM HTL-Atto655 (red), respectively. (B) SpaS-SNAP-tag and FliN-HaloTag were stained with 30 nM TMR-Star (green) and 150 nM HTL-Atto655 (red), respectively. After staining, cells were washed and fixed with 3% PFA and immobilized on glass slides. For dSTORM imaging, cells were incubated in a buffer containing 100 mM β-Mercaptoethylamine, 4.5 mg × ml⁻¹ D-Glucose, 40 μg × ml⁻¹ Catalase and 0.5 mg × ml⁻¹ Glucose-Oxidase and maximum laser power was used for excitation. SR images were rendered from single emitter localizations obtained within 500 frames. SR images of the TMR channel (i), of the Atto655 channel (ii) and merged images of both channels (iii) are shown. For the shape of the bacteria, the maximum intensity projection with bilinear interpolation of all 500 acquired frames is shown in the lower corner in (iii). Scale bar, 0.5 μm.