Figure 3: Labelling specificity probed by single molecule localization.

(A) MvP818 [p3725], the invC deletion strain expressing invC::HaloTag, was treated with 20 nM HTL-TMR or 150 mM HTL-Atto655. MvP818 [p3723], the invC deletion strain expressing invC::SNAP-tag, was treated with 30 nM TMR-Star. Labeling reactions were performed for 45 min at 37 °C. For the shape of the bacteria, the maximum intensity projection with bilinear interpolation of all 500 acquired frames is shown (left panel). After localization distinct patches of InvC are visible for each condition. The white dots in the localized images show the localized molecules of all 500 acquired frames (right panel). Clusters are indicated with red arrows. (B) Salmonella WT cells were treated with 20 nM HTL-TMR, 150 nM HTL-Atto655 or 30 nM TMR-Star for 45 min at 37 °C. 500 frames were acquired as described before with 5 mW laser power at the focal plane. The autofluorescence of bacteria upon excitation with a 488 nm laser is shown (left panel). SRM images rendered after localization of individual emitters (right panel) confirmed labelling specificity in all cases. Scale bars, 1 μm.