A general one-step protocol to generate impermeable fluorescent HaloTag substrates for in situ live cell application...

Visualization of proteins can be achieved by genetically grafting HaloTag Protein (HTP) into the protein of interest followed by incubation with a dye-linked HaloTag Ligand (HTL). This approach allows for use of fluorophores optimized for specific optical techniques or of cell-impermeable dyes to selectively label cell surface proteins. However, these two goals often conflict, as many high-performing dyes exhibit membrane permeability. Here we show that several dye-HTL reagents can be made cell-impermeable by inserting a charged sulfonate directly into the HTL, leaving the dye moiety unperturbed, using a one-step protocol. We validate such compounds, termed dye-SHTL (dye shuttle), in living cells, and demonstrate exclusive membrane staining. In transduced primary hippocampal neurons, we label a neuromodulatory receptor with dyes optimized for stimulated emission by depletion super-resolution microscopy, allowing accuracy in distinguishing surface versus internal receptors of the presynaptic terminal. This approach offers broad utility for surface-specific protein labelling.