A general one-step protocol to generate impermeable fluorescent HaloTag substrates for in situ live cell application and super-resolution imaging
- Kilian Roßmann
- Ulrich Pabst
- Bianca C. Baciu
- Siqi Sun
- Christiane Huhn
- Christina Holmboe Olesen
- Maria Kowald
- Eleni Tapp
- Marie Bieck
- Ramona Birke
- Brenda C. Shields
- Pyeonghwa Jeong
- Jiyong Hong
- Michael R. Tadross
- Joshua Levitz
- Martin Lehmann
- Noa Lipstein
- Johannes Broichhagen
2026-01-12
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.