Figure 1: Logic for impermeable dyes to label cell surface proteins.

A-B) Known strategies to address extracellularly exposed self-labelling tags with cartoons and chemical structures indicating where anionic charges have previously been introduced. C-D) Our new strategy to install a sulfonate charge on the HaloTag Ligand (HTL). E) Modelling of the HaloTag Protein (HTP) bound to TMR-SHTL. F) Synthesis of TMR-d12-SHTL and SiR-d12-SHTL. G) Excitation and emission profiles of TMR-d12 comparing HTL to SHTL conjugates. H) In vitro protein labelling of apo-HTP confirms binding by full protein mass spectrometry. I) Labelling kinetics of apo-HTP with TMR-d12-HTL and TMR-d12-SHTL. J) Fluorescence quantum yields of TMR/SiR-d12 conjugated to HTL/SHTL with or without HTP-bound. K) Fluorescence lifetimes of the same reagents. L) Table summarizing values from G, J, K.