No-wash live cell imaging of HaloTag-expressing cells with a Si-Bridge dye. The fluorogenic behavior of 68b for DNA labeling suggested that fluorogenic probes for other valuable classes of live cell targets could be developed. Halo-Tag presents an anionic surface that is known to favor spirolactone dye ring-opening. Our operating hypothesis is that favorable biomolecular interaction with the ring-opened cationic dye will occur only when the anionic carboxylate is facing away from the protein surface (Figure ). Thus, we predicted that fluorogenic Si-Bridge dyes targeting HaloTag will also favor r,r isomer b, with the Si-tether and the carboxylate on opposite faces of the dye. To test this supposition, we transiently transfected HeLa cells with pHaloTag-EGFP and treated the cells with 200 nM of six Si-Bridge dyes modified with chloroalkane ligands for HaloTag (70, 71a, 71b, 75a, 75b, and 76b in Figure ). As anticipated, the r,r isomer 75b and the corresponding azetidine analog 76b are fluorogenic and labeling correlates with GFP expression (Figure , Figure ). Dye 71b also labeled HaloTag-EGFPexpressing cells to some extent but had significant background (Figure ), suggesting that cationic dye 70 only stained mitochondria, suggesting that sequestration by the negatively polarized mitochondrial membrane dominates its behavior in cells (Figure ).